Medicine for improving state of pregnancy, and use thereof

ABSTRACT

A medicine for improving the state of pregnancy, which comprises a compound represented by general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient; and a use of the medicine.

TECHNICAL FIELD

The present invention relates to a medicine or a medical agent forameliorating pregnancy conditions including sterility and/orinfertility, the medicine including a particular immunosuppressivecomponent, and utilization of the medicine.

BACKGROUND ART

There are a large number of people who have finally given up bearingchildren even though they hope for bearing children, because they areincapable of normal pregnancy or childbearing. When the condition ofbeing incapable of normal pregnancy or childbearing is categorized bystages, the condition can be divided into a condition in whichimplantation of a fertilized ovum into the endometrium does not proceedproperly, and pregnancy is not established (sterility in a broad sense);and a condition in which implantation of a fertilized ovum into theendometrium proceeds properly, but a child is not born due tomiscarriage, stillbirth, and the like (infertility in a broad sense).

Sterility and infertility may be considered as subjects for treatment asso-called infecundity and infertilitas, when the condition sets in.Under the current circumstances, for the treatment of infecundity andinfertilitas, therapeutic methods that are considered optimal areselected according to the causes. For example, in the case ofinfecundity and infertilitas caused by blood coagulation disorder,diagnostic methods and therapeutic indicators are available, and thetherapeutic methods have been almost established. However, for thereason that infecundity and infertilitas without clearly specifiedcauses also exist at a considerable proportion, clinicians often need tostruggle to find proper treatments. For example, under severeinfecundity, there exist cases with disorder of implantation, in whichimpregnation fails even though treatment based on in vitro fertilizationis attempted several times. Under severe infertilitas, there existnumerous cases in which subjects are resistant to the therapy forinfertilitas that is currently implemented. There are no specialtherapeutic methods in connection with severe infecundity that isconsidered cryptogenic; however, in connection with severe infertilitas,for example, antiplatelet therapy (administration of low-dose aspirin),anticoagulation therapy (administration of heparin), steroid therapy(PSL; prednisolone), and large-quantity gamma-globulin therapy (IVIG)may be carried out.

CITATION LIST Non Patent Literature

Non Patent Literature 1: Uemura Y, Suzuki M, Liu T Y, Narita Y, HirataS, Ohyama H, Ishihara O., Matsushita S. Int Immunol. 2008; 20: 405-12.

Non Patent Literature 2: Saito S, Nakashima A, Shima T, Ito M. Am JReprod Immunol. 2010; 63: 601-10.

Non Patent Literature 3: Kwak-Kim J Y, Chung-Bang H S, Ng S C, NtrivalasE I, Mangubat C P, Beaman K D, Beer A E, Gilman-Sachs A. Hum Reprod.2003; 18: 767-73.

Non Patent Literature 4: Ng S C, Gilman-Sachs A, Thaker P, Beaman K D,Beer A E, Kwak-Kim J. Am J Reprod Immunol. 2002 Aug; 48 (2): 77-86.

Non Patent Literature 5: Yamaguchi K, Hisano M, Isojima S, Irie S, ArataN, Watanabe N, Kubo T, Kato T, Murashima A. J Med Virol. 2009; 81:1923-8.

SUMMARY OF INVENTION Technical Problem

A fertilized ovum or a fetus is a foreign material (a kind of exogenousantigen) to the mother's body. Therefore, it is considered that duringthe period extending from the implantation of a fertilized ovum andmaintenance of pregnancy to the childbirth, changes occur in the immunemechanism of the mother's body in order for the mother's body tocontinue accepting the fertilized ovum or the fetus without eliminationfrom the mother's body. For example, it is considered that in theintrauterine decidua, it is one important factor for continuingpregnancy to promote the suppression of the immune mechanism centered oncell-mediated immunity in order to prevent exclusion of the fertilizedovum or the fetus as a foreign material (Non Patent Literatures 1 to 5).However, the immune mechanism is very complicated, and the details ofthe changes occurring in the immune mechanism of the mother's bodyduring the period from the implantation of the fertilized ovum tochildbirth still need to be clarified in large part.

Under such circumstances, among existing therapeutic methods,immunosuppressive therapy for the treatment of infecundity does notexist.

For the treatment of infertilitas, it is considered that the steroid(PSL) therapy or large-quantity gamma-globulin therapy (IVIG) can actsuppressively on the immune mechanism. However, it has been reportedthat either therapeutic method has various undesirable influences on themother's body and the fetus. Furthermore, IVIG therapy also has aproblem that there is a risk of unknown infection attributed to the useof a blood plasma fractionation (purified from pooled plasma of 1,000 ormore persons), and the operating period is short.

However, it has been gradually clarified by preliminary search of theinventors of the present invention that among the patients ofinfecundity, there are people who are suspected to undergo anenhancement in the cell-mediated immunity compared to the state beforepregnancy and have a rejection reaction against the fertilized ovum atthe time of implantation; and among the patients of infertilitas, thereare people who do not have abnormalities in the immune state beforeimplantation, but are suspected to undergo a noticeable enhancement inthe cell-mediated immunity after implantation (after establishment ofpregnancy) and thereby have a rejection reaction against the fetus. Inboth cases, it is speculated that having insufficient suppressivemechanism for immunity that attacks the fertilized ovum or the fetus(immunological tolerance) is an important factor.

Based on these findings, the inventors of the present invention presumedthat reception of a fertilized ovum or an embryo having an isoantigenonto the endometrium could result in an immunoresponse that is verysimilar to organ transplantation, and attempted to verify thispresumption from various angles. As a result, the inventors found amedicine that exhibits an effect of sufficiently ameliorating theconditions of sterility or infertility and with which any undesirableinfluence on the mother's body or the fetus is suppressed, and thusconceived the present invention. The inventors also found that thismedicine may also be helpful for the amelioration of pregnancyconditions other than sterility or infertility, and thus the inventorsconceived the present invention.

Solution to Problem

In order to solve the problems described above, the present inventionincludes the following embodiments.

A medicine for ameliorating pregnancy conditions, the medicine includinga compound represented by the following General Formula (I) or apharmaceutically acceptable salt thereof as an active ingredient:

wherein each of the adjacent pairs of R¹ with R², R³ with R⁴, and R⁵with R⁶ independently

(a) represents two adjacent hydrogen atoms, or R² may be an alkyl group,or

(b) may form another bond between the carbon atoms to which the pairmembers are respectively bonded;

R⁷ represents a hydrogen atom, a hydroxyl group, or a protected hydroxylgroup, or may be bonded to an alkyl and together represent an oxo group;

R⁸ and R⁹ each independently represent a hydrogen atom or a hydroxylgroup;

R¹⁰ represents a hydrogen atom, an alkyl group, an alkyl groupsubstituted with one or more hydroxyl groups, an alkenyl group, analkenyl group substituted with one or more hydroxyl groups, or an alkylgroup substituted with an oxo group;

X represents an oxo group, (a hydrogen atom, a hydroxyl group), (ahydrogen atom, a hydrogen atom), or a group represented by formula:—CH₂O—;

Y represents an oxo group, (a hydrogen atom, a hydroxyl group), (ahydrogen atom, a hydrogen atom), or a group represented by formula:N—NR¹¹R¹²or formula: N—OR¹³;

R¹¹ and R¹² each independently represent a hydrogen atom, an alkylgroup, an aryl group, or a tosyl group;

R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²², and R²³ each independentlyrepresent a hydrogen atom or an alkyl group;

R²⁴ represents a ring which can include one or more heteroatoms and maybe substituted as desired; and

n represents 1 or 2, and

in addition to the meanings described above, Y, R¹⁰ and R²³ may also bebonded together with the carbon atoms to which Y, R¹⁰ and R²³ arebonded, and represent a heterocyclic group formed from a saturated orunsaturated 5-membered or 6-membered ring and containing a nitrogenatom, a sulfur atom, and/or an oxygen atom, and the heterocyclic groupmay be substituted with one or more groups selected from an alkyl group,a hydroxyl group, an alkyloxy group, a benzyl group, a group representedby formula: —CH₂Se(C₆H₅), and an alkyl group substituted with one ormore hydroxyl groups.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, pregnancy conditions includingsterility and/or infertility can be ameliorated.

DESCRIPTION OF EMBODIMENTS

Hereinafter, the present invention will be explained in detail.

1. Medicine (Pharmaceutical Composition) Active Ingredient

The medicine according to the present invention includes, as an activeingredient, a compound represented by the following General Formula (I)(hereinafter, these may be collectively referred to as Compound (I)) ora pharmaceutically acceptable salt thereof as an active ingredient.

Compound (I) is a macrolide-based compound. Macrolide-based compound isa generic name for compounds which are large macrocyclic lactones andhave 12 or more ring member atoms.

wherein each of the adjacent pairs of R¹ with R², R³ with R⁴, and R⁵with R⁶ independently:

(a) represents two adjacent hydrogen atoms, or R² may be an alkyl group,or

(b) may form another bond between the carbon atoms to which the pairmembers are respectively bonded;

R⁷ represents a hydrogen atom, a hydroxyl group, or a protected hydroxylgroup, or may be bonded to an alkyl and together represent an oxo group;

R⁸ and R⁹ each independently represent a hydrogen atom or a hydroxylgroup;

R¹⁰ represents a hydrogen atom, an alkyl group, an alkyl groupsubstituted with one or more hydroxyl groups, an alkenyl group, analkenyl group substituted with one or more hydroxyl groups, or an alkylgroup substituted with an oxo group;

X represents an oxo group, (a hydrogen atom, a hydroxyl group), (ahydrogen atom, a hydrogen atom), or a group represented by formula:—CH₂O—;

Y represents an oxo group, (a hydrogen atom, a hydroxyl group), (ahydrogen atom, a hydrogen atom), or a group represented by formula:N—NR¹¹R¹²or formula: N—OR¹³;

R¹¹ and R¹² each independently represent a hydrogen atom, an alkylgroup, an aryl group, or a tosyl group;

R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²², and R²³ each independentlyrepresent a hydrogen atom or an alkyl group;

R²⁴ represents a ring which can include one or more heteroatoms and maybe substituted as desired; and

n represents 1 or 2.

In addition to the meanings described above, Y, R¹⁰ and R²³ may also bebonded together with the carbon atoms to which Y, R¹⁰ and R²³ arebonded, and represent a heterocyclic group formed from a saturated orunsaturated 5-membered or 6-membered ring and containing a nitrogenatom, a sulfur atom, and/or an oxygen atom, and the heterocyclic groupmay be substituted with one or more groups selected from an alkyl group,a hydroxyl group, an alkyloxy group, a benzyl group, a group representedby formula: —CH₂Se(C₆H₅), and an alkyl group substituted with one ormore hydroxyl groups.

Preferable R²⁴ may be a cyclo-(C₅₋₇) alkyl group which may have anappropriate substituent, and examples thereof include, for example, thefollowing groups.

(a) a 3,4-dioxo-cyclohexyl group;

(b) a 3-R²⁰-4-R²¹-cyclohexyl group, wherein R²⁰ represents a hydroxyl,an alkyloxy, an oxo, or OCH₂OCH₂CH₂OCH₃, and R²¹ represents a hydroxyl,—OCN, an alkyloxy, a heteroaryloxy which may have an appropriatesubstituent, —OCH₂OCH₂CH₂OCH₃, a protected hydroxyl, a chloro, a bromo,an iodo, an aminooxalyloxy, an azide group, a p-tolyloxythiocarbonyloxy,or R²⁵R²⁶CHCOO—(wherein R²⁵ represents a hydroxyl group which may beprotected as desired, or a protected amino group; and R²⁶ represents ahydrogen atom or a methyl, or R²⁰ and R²¹ may be bonded together andform an oxygen atom of an epoxide ring); or

(c) a cyclopentyl group, the cyclopentyl group being substituted with amethoxymethyl, a hydroxymethyl protected as desired, an acyloxymethyl(wherein the acyl moiety is a dimethylamino group which may bequaternized as desired, or a carboxyl group which may be esterified asdesired), one or more amino and/or hydroxyl groups which may beprotected, or an aminooxalyloxymethyl. A preferred example is a2-formylcyclopentyl group.

The various definitions and specific examples thereof used for GeneralFormula (I), and preferred embodiments thereof will be explained indetail below.

Unless particularly stated otherwise, the term “lower” is intended tomean a group having 1 to 6 carbon atoms.

A preferred example of the alkyl moiety of the “alkyl group” and the“alkyloxy group” may be a linear or branched aliphatic hydrocarbonresidue, and examples include lower alkyl groups such as methyl, ethyl,propyl, isopropyl, butyl, isobutyl, pentyl, neopentyl, and hexyl.

A preferred example of the “alkenyl group” may be a linear or branchedaliphatic hydrocarbon residue containing one double bond, and examplesinclude lower alkenyl groups such as vinyl, propenyl (allyl or thelike), butenyl, methylpropenyl, pentenyl, and hexenyl.

Preferred examples of the “aryl group” include phenyl, tolyl, xylyl,cumenyl, mesityl, and naphthyl.

Preferred protective groups for the “protected hydroxyl group” and the“protected amino” include, for example, 1-(lower alkylthio) (lower)alkyl groups such as a lower alkylthiomethyl groups, such asmethylthiomethyl, ethylthiomethyl, propylthiomethyl,isopropylthiomethyl, butylthiomethyl, isobutylthiomethyl, orhexylthiomethyl, with a more preferred example being a C₁-C₄alkylthiomethyl group, and the most preferred example being amethylthiomethyl group;

trisubstituted silyl groups, for example, a tri(lower) alkylsilyl suchas trimethylsilyl, triethylsilyl, tributylsilyl, tertiarybutyl-dimethylsilyl, or tri-tertiary butylsilyl, and for example, alower alkyldiarylsilyl such as methyldiphenylsilyl, ethyldiphenylsilyl,propyldiphenylsilyl, or tertiary butyldiphenylsilyl, more preferredexamples being a tri(C₁-C₄) alkylsilyl group and a C₁-C₄alkyldiphenylsilyl group, and the most preferred examples being atertiary butyl-dimethylsilyl group and a tertiary butyldiphenylsilylgroup; and

acyl groups such as an aliphatic acyl group derived from a carboxylicacid, a sulfonic acid or a carbamic acid, an aromatic acyl group, and analiphatic acyl group substituted with an aromatic group.

Examples of the aliphatic acyl group include a lower alkanoyl groupwhich may have one or more appropriate substituents such as a carboxyl,such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl,isovaleryl, pivaloyl, hexanoyl, carboxyacetyl, carboxypropionyl,carboxybutyryl, or carboxyhexanoyl;

a cyclo-(lower) alkyloxy-(lower) alkanoyl group which may have one ormore appropriate substituents such as a lower alkyl, such ascyclopropyloxyacetyl, cyclobutyloxypropionyl, cycloheptyloxybutyryl,menthyloxyacetyl, menthyloxypropionyl, menthyloxybutyryl,menthyloxypentanoyl, or a menthyloxyhexanoyl;

a camphorsulfonyl group; and

a lower alkylcarbamoyl group having one or more appropriate substituentssuch as a carboxyl or a protected carboxyl, for example, acarboxy-(lower) alkylcarbamoyl group such as carboxymethylcarbamoyl,carboxyethylcarbamoyl, carboxypropylcarbamoyl, carboxybutylcarbamoyl,carboxypentylcarbamoyl or carboxyhexylcarbamoyl, or a tri-(lower)alkylsilyl-(lower) alkyloxycarbonyl-(lower) alkylcarbamoyl group such astrimethylsilylmethoxycarbonylethylcarbamoyl,trimethylsilylethoxycarbonylpropylcarbamoyl,triethylsilylethoxycarbonylpropylcarbamoyl, tertiarybutyldimethylsilylethoxycarbonylpropylcarbamoyl, ortrimethylsilylpropoxycarbonylbutylcarbamoyl group.

Examples of the aromatic acyl group include, an aroyl group which mayhave one or more appropriate substituents such as a nitro, for example,benzoyl, toluoyl, xyloyl, naphthoyl, nitrobenzoyl, dinitrobenzoyl, ornitronaphthoyl; and

an arenesulfonyl group which may have one or more appropriatesubstituents such as a halogen, for example, benzenesulfonyl,toluenesulfonyl, xylenesulfonyl, naphthalenesulfonyl,fluorobenzenesulfonyl, chlorobenzenesulfonyl, bromobenzenesulfonyl, oriodobenzenesulfonyl.

The aliphatic acyl group substituted with an aromatic group may be, forexample, an ar-(lower) alkanoyl group which may have one or moreappropriate substituents such as a lower alkyloxy or a trihalo-(lower)alkyl, for example, phenylacetyl, phenylpropionyl, phenylbutyryl,2-trifluoromethyl-2-methoxy-2-phenylacetyl,2-ethyl-2-trifluoromethyl-2-phenylacetyl, or2-trifluoromethyl-2-propoxy-2-phenylacetyl.

Among the acyl groups described above, more preferred acyl groupsinclude a C₁-C₄ alkanoyl group which may have a carboxyl, acyclo-(C₅-C₆) alkyloxy-(C₁-C₄) alkanoyl group having two (C₁-C₄) alkylsin the cycloalkyl moiety, a camphorsulfonyl group, a carboxy-(C₁-C₄)alkylcarbamoyl group, a tri-(C₁-C₄) alkylsilyl-(C₁-C₄)alkyloxycarbonyl-(C₁-C₄) alkylcarbamoyl group, a benzoyl group which mayhave one or two nitro groups, a benzenesulfonyl group having a halogen,and a phenyl-(C₁-C₄) alkanoyl group having a C₁-C₄ alkyloxy and atrihalo-(C₁-C₄) alkyl. Among them, the most preferred examples includeacetyl, carboxypropionyl, menthyloxyacetyl, camphorsulfonyl, benzoyl,nitrobenzoyl, dinitrobenzoyl, iodobenzenesulfonyl, and2-trifluoromethyl-2-methoxy-2-phenylacetyl.

Preferred examples of the “heterocyclic group formed from a saturated orunsaturated 5-membered or 6-membered ring and containing a nitrogenatom, a sulfur atom, and/or an oxygen atom” include a pyrrolyl group anda tetrahydrofuryl group.

Regarding the “heteroaryl moiety which may have an appropriatesubstituent” in the “heteroaryloxy which may have an appropriatesubstituent”, the moieties listed as examples of group R1 of thecompound represented by the formula in EP-A-532,088 may be mentioned;however, for example, a 1-hydroxyethylindol-5-yl is preferred. Thedisclosure of the patent literature is partially incorporated herein byreference.

It is described in, for example, EP-A-184162, EP-A-323042, EP-A-423714,EP-A-427680, EP-A-465426, EP-A-480623, EP-A-532088, EP-A-532089,EP-A-569337, EP-A-626385, WO 89/05303, WO 93/05058, WO 96/31514, WO91/13889, WO 91/19495, and WO 93/5059 that the compound (I) and apharmaceutically acceptable salt thereof used for the present inventionhave excellent immunosuppressive action, antibacterial activity, andother pharmacological activity, and therefore, the compounds are usefulfor the treatment and prevention of a rejection reaction against thetransplantation of an organ or a tissue, a graft-versus-host reaction,an autoimmune disease, an infectious disease, and the like. Furthermore,production methods for those compounds are also disclosed therein. Thedisclosures are partially incorporated herein by reference.

Particularly, compounds called FR900506 (=FK506, tacrolimus), FR900520(ascomycin), FR900523 and FR900525 are substances produced by the genusStreptomyces, for example, Streptomyces tsukubaensis No. 9993(depository: National Institute of Bioscience and Human-TechnologyAgency of Industrial Science and Technology, the Ministry ofInternational Trade and Industry, 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan (formerly: Fermentation Research Institute, Agency ofIndustry Science and Technology, the Ministry of International Trade andIndustry), date of deposit: Oct. 5, 1984, deposit number: FERM BP-927)or Streptomyces hygroscopicus subsp. yakushmaensis No. 7238 (depository:National Institute of Bioscience and Human-Technology Agency ofIndustrial Science and Technology, the Ministry of International Tradeand Industry, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan,date of deposit: Jan. 12, 1985, deposit number: FERM BP-928)(EP-A-0184162). Particularly, FK506 (general name: tacrolimus)represented by the following structural formula is a representativecompound.

Chemical name:

17 -Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone.

Among the examples of Compound (I), more preferred is a compound inwhich each of the adjacent pairs of R³ with R⁴ and R⁵ with R⁶ formsanother bond between the carbon atoms to which the pair members arerespectively bonded;

R⁸ and R²³ each independently represent a hydrogen atom;

R⁹ represents a hydroxyl group; R¹⁰ represents a methyl, ethyl, propyl,or allyl group;

X represents (a hydrogen atom, a hydrogen atom) or an oxo group;

Y represents an oxo group;

R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, and R²² each represent a methyl group;

R²⁴ represents a 3-R²⁰-4-R²¹ -cyclohexyl group (wherein R²⁰ represents ahydroxyl, an alkyloxy, an oxo, or —OCH₂OCH₂CH₂OCH₃; and R²¹ represents ahydroxyl, —OCN, an alkyloxy, a heteroaryloxy which may have anappropriate substituent, a 1-tetrazolyl or a 2-tetrazolyl,—OCH₂OCH₂CH₂OCH₃, a protected hydroxyl, a chloro, a bromo, an iodo, anaminooxalyloxy, an azide group, a p-tolyloxythiocarbonyloxy, orR²⁵R²⁶CHCOO— (wherein R²⁵ represents a hydroxyl group which may beprotected as desired, or a protected amino group; and R²⁶ represents ahydrogen atom or a methyl), or

R²⁰ and R²¹ may be bonded together and form an oxygen atom of an epoxidering; and

n represents 1 or 2.

Preferred examples of Compound (I) include tacrolimus, and ascomycin ora derivative thereof, for example, 33-epi-chloro-33-desoxyascomyindescribed in Example 66a of EP 427,680. Other preferred examples ofCompound (I) include, for example, the compound described in Example 6dof EP 569,337 and the compound described in Example 8 of EP 626,385.

The compounds described in EP 0184162, EP 323042, EP 424714, EP 427680,EP 465426, EP 474126, EP 480623, EP 484936, EP 532088, EP 532089, EP569337, EP 626385, WO 89/05303, WO 93/05058, WO 96/31514, WO 91/13889,WO 91/19495, WO 93/5059, WO 96/31514 and the like may also be mentionedas preferred examples of the macrolide-based Compound (I), and thedisclosures of the documents are partially incorporated herein byreference.

According to another embodiment of the present invention, the medicineaccording to the present invention includes, as an active ingredient,the compound shown below or a pharmaceutically acceptable salt thereof.

Regarding the compound that acts as an active ingredient for themedicine of the present invention, cyclosporins, for example,cyclosporin A, B, D and the like, which are used instead of Compound (I)or a salt thereof, or used in combination with Compound (I) or a saltthereof. These are described in Merck Index (12^(th) edition), No. 2821,the disclosure of which is partially incorporated herein by reference.

A preferred macrolide-based compound that is used as an activeingredient instead of Compound (I) or a salt thereof, or used as anactive ingredient in combination with Compound (I) or a salt thereof,for the medicine of the present invention may be rapamycin described inMerck Index (12^(th) edition), No. 8288, or a derivative thereof.Preferred examples thereof include O-substituted derivatives in whichthe hydroxyl at the 40-position of Formula A in page 1 of WO 95/16691 issubstituted by —OR¹ (wherein R¹ represents a hydroxyalkyl, ahydroalkyloxyalkyl, an acylaminoalkyl, or an aminoalkyl), for example,40-O-(2-hydroxy)ethyl-rapamycin, 40-O -(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, and40-O-(2-acetaminoethyl)-rapamycin.

These O-substituted derivatives can be produced by a reaction betweenrapamycin (or dihydro- or deoxorapamycin) and an organic radical bondedto a leaving group (for example, RX (wherein R represents an organicradical that is desirable as an O-substituent such as an alkyl, allyl orbenzyl moiety; and X represents a leaving group such as CC₁₃C(NH)O orCF3SO₃).

Regarding the conditions, when X represents CC₁₃C(NH)O, the conditionsmay be acidic or neutral conditions, for example, in the presence oftrifluoromethanesulfonic acid, camphorsulfonic acid, p-toluenesulfonicacid, or a pyridinium or substituted pyridinium salt corresponding tosuch an acid; or when X represents CF3SO₃, the conditions may be in thepresence of a base such as pyridine, a substituted pyridine,diisopropylethylamine, or pentamethylpiperidine. The most preferredrapamycin derivative is 40-O-(2-hydroxy)ethyl-rapamycin as described inWO 94/09010, the disclosure of which is partially incorporated herein byreference.

Compound (I), rapamycin, and derivatives thereof have similar basicskeletons, namely, tricyclomacrolide skeletons, and at least one similarbiological characteristic (for example, immunosuppressive action).

The pharmaceutically acceptable salts of Compound (I), cyclosporin,rapamycin, and derivatives thereof are pharmaceutically acceptablenon-toxic salts that are conventionally used, and examples thereofinclude salts with inorganic or organic bases, such as alkali metalsalts such as sodium salts and potassium salts; for example, alkalineearth metal salts such as calcium salts and magnesium salts; ammoniumsalts; and for example, amine salts such as triethylamine salts andN-benzyl-N-methylamine salts.

Compound (I) of the present invention may have conformers, or one ormore pairs of stereoisomers such as optical isomers and geometricisomers attributed to asymmetric carbon atoms and double bonds. Thoseconformers and isomers are also included in the scope of the compound ofthe present invention.

Furthermore, Compound (I) may also form a solvate, and in that case, thesolvate is also included in the scope of the present invention.Preferred solvates include hydrate and ethanolate.

Other Optional Components

The medicine according to the present invention may also include, inaddition to the active ingredient described above, one or moretherapeutically active substances having therapeutic action on otherdiseases, illnesses and conditions, as long as the therapeuticallyactive substances have no risk of inhibiting the activity of the activeingredient and are not harmful to the subject for administration(hereinafter, also referred to as patient).

Furthermore, the medicine of the present invention may also include apharmaceutically acceptable carrier that is not harmful to the subjectfor administration. The carrier that can be used may be any of a solidcarrier, a semisolid carrier, and a liquid carrier, and may be, forexample, any one selected from water, a liquid electrolyte, and aglucose solution, without any particular limitations. The therapeuticmedicine may also include auxiliary agents. Examples of the auxiliaryagents include a lubricating agent, a stabilizer, an antiseptic agent,an emulsifier, a thickener (viscous agent), a colorant, a fragrance(flavoring agent), an excipient, a preservative, a buffering agent, acorrigent, a suspending agent, an emulsifier, a dissolution aid, and aflow conditioner.

Dosage Form

Examples of the dosage form include a tablet, a capsule, a pill, agranular preparation, a powder, a syrup, a suppository, a troche, apellet, an emulsion, a suspension, and other known forms. Among these,for example, the dosage form as a preparation for oral administration ispreferably any of a tablet, a capsule, a pill, a granular preparation, apowder, a liquid, a syrup, and a jelly; more preferably any of a tablet,a capsule, and a granular preparation; and even more preferably atablet. As will be described below, the medicine may be formulated as apreparation for parenteral administration such as, for example, aninjectable preparation, a suppository, and a percutaneous absorptiontype preparation.

Method for Producing Medicine

The medicine according to the present invention can be produced byutilizing any known production method. For example, the medicine isproduced by preparing an active ingredient and other optional componentsseparately for each component, and then mixing the respective componentsso as to obtain desired contents.

Subject for Administration for Medicine Subject Biological Species

The subject for administration of the medicine of the present inventionmay include a mammal, a bird, a reptile, an amphibian, and the like, andamong them, a mammal is preferred. Examples of the mammal include ahuman being and animals other than human being, including livestock suchas cattle, horse, pig, and sheep, and pets such as dog, cat, rat, andrabbit. A preferred subject for administration is a human being.Examples of the bird include poultry such as chicken, wild duck, anddomestic duck.

Defective Pregnancy Conditions

The subject for administration of the medicine of the present inventionis the above-described subject with defective pregnancy conditions.Here, the term “defective pregnancy conditions” is a concept categorizedas an illness or a disease; however, in addition to that, the term is aconcept that also includes a state that is defective compared to normalpregnancy. That is, defective pregnancy conditions include thedefectiveness of conception in the mother's body and the fetal state, aswell as the defectiveness in the health state of the mother's bodyitself during pregnancy. The term “during pregnancy” refers to anarbitrary period during the term from establishment of pregnancy(synonym for achievement of pregnancy) to childbirth, and includes thewhole period of the first trimester of pregnancy, the second trimesterof pregnancy, and the third trimester of pregnancy. Here, the firsttrimester of pregnancy refers to the period from the establishment ofpregnancy to the thirteenth week; the second trimester of pregnancyrefers to the period from the 14^(th) week to the 27^(th) week; and thethird trimester of pregnancy refers to the period from the 28^(th) weekto the day of delivery. Furthermore, defective pregnancy conditionsinclude defectiveness in the health state of the mother's body afterchildbirth, and the health state of the mother's body during pregnancyand after delivery, which appears as disorders in the offspring afterbirth.

The type of defective pregnancy conditions is not particularly limited;however, specific examples include at least one selected from (1)sterility, (2) infertility, and (3) defective pregnancy conditions otherthan sterility and infertility (for example, pregnancy complications).Among these, as will be described below, a defective pregnancy conditionattributed to immunological abnormalities of the subject foradministration is more suitable as the defectiveness to be considered asan object of amelioration by administration of the medicine of thepresent invention.

Sterility and Infertility

The term “sterility” according to the present specification is used as ameaning in a broad sense, and refers to a state in which the mother'sbody has difficulties in becoming pregnant compared to the normal state.Sterility is a concept including infecundity. Here, infecundity refersto the case in which pregnancy is not established within one yearwithout contraception under an expectation of childbearing.

According to an embodiment, the subject with sterility, who becomes thesubject for administration of the medicine of the present invention, maybe receiving an existing treatment for infecundity. Examples of themethod for treating such infecundity include artificial insemination, invitro fertilization, and administration of a fertility medicine.

Among these, specific methods for in vitro fertilization include methodssuch as embryo transplantation, by which collected ova and sperms arefertilized in a Petri dish, and fertilized ova are transferred back intothe womb (In Vitro Fertilization Embryo Transfer: IVF-ET), andIntracytoplasmic Sperm Injection (ICSI), by which fertilization isperformed by directly injecting a sperm into an ovum under a microscope.Further examples of IVF-ET include fresh embryo transplantation (freshET) of using a fresh embryo for transplantation, and freeze-thawedembryo transplantation (FET) of freezing and thawing an embryo that hasbeen preserved in a frozen state after collection and then using theembryo for transplantation. The subject may be any person who is unableto have pregnancy established (a clear increase in the hCG level is notobserved) even if embryo transplantation or intracytoplasmic sperminjection is carried out several times, and the techniques can also besuitably applied to a person who is unable to have pregnancy establishedeven if treatment is carried out three or more times, four or moretimes, or five or more times.

Causes for sterility including infecundity include an ovum factor, anoviduct factor, a uterine factor, a cervical duct factor, and animmunological factor. Specific examples of the ovum factor includeovulation disorders caused by antiprolactinemia, polycystic ovarysyndrome, various stresses, dieting, and premature ovarian dysfunction.Specific examples of the oviduct factor include blockage, stenosis oradhesion of the Fallopian tubes caused by Chladimyal salpingitis andperisalpingitis, and the like. Specific examples of the uterine factorinclude uterine myoma, endometrial polyp, congenital anomaly of uterus,and intrauterine adhesion (Asherman's syndrome). Specific examples ofthe cervical duct factor include cervicitis, and abnormal mucussecretion or impaired mucus secretion from the cervical duct. Specificexamples of the immunological factor include production of anti-spermantibodies. Here, sterility of unknown cause implies that all of theabove-mentioned factors do not apply, and the cause is not specified. Itis considered that sterility of unknown cause includes sterilityattributed to immunological abnormalities (including abnormalities inimmune cells, and autoimmune diseases such as anti-phospholipid antibodysyndrome), and a subject with such sterility of unknown cause is alsosuitable as a subject for administration of the medicine of the presentinvention (also see Examples). Particularly, in the case of having anautoimmune disease or in the case of not having an autoimmune disease, asubject with sterility that is presumed to be primarily caused byinhibition of implantation of a fertilized ovum or an embryo into theendometrium due to immunological abnormalities, is suitable as a subjectfor administration of the medicine of the present invention.

The term “infertility” according to the present specification refers toa state in which after conception, the fetus in the womb of the mother'sbody is incapable of growing, or a state in which retarded growth ordefective growth of the fetus is observed, and the term is a conceptincluding infertilitas. Here, infertilitas refers to the case in whichpregnancy (also including spontaneous pregnancy as well as the cases ofartificial fertilization and in vitro fertilization) is established, butmiscarriage or stillbirth is repeated two or more times so thatchildbearing is not achieved. Meanwhile, experiencing miscarriage,premature delivery, or stillbirth once, or repeating miscarriage,premature delivery, or stillbirth two times is also included in theconcept of “infertility” according to the present invention.

Causes for infertility including infertilitas include a genetic factor,an anatomic factor, an endocrine factor, a coagulative factor, and anautoimmunologic factor. Specific examples of the genetic factor includechromosome aberration of parents, and fetal chromosome aberration.Specific examples of the anatomic factor include myoma and uterinemalformation. Specific examples of the endocrine factor include corpusluteum insufficiency, hyperprolactinemia, thyroid dysfunction, and bloodsugar abnormalities. Specific examples of the coagulative factor includeblood coagulation abnormalities. Specific examples of theautoimmunologic factor include production of anti-phospholipidantibodies (anti-phospholipid antibody syndrome). Here, infertility ofunknown cause implies that all of the above-mentioned factors do notapply, and the cause is not specified. It is considered that infertilityof unknown cause includes infertility attributed to immunologicalabnormalities (including abnormalities in immune cells, and autoimmunediseases such as anti-phospholipid antibody syndrome), and a subjectwith such infertility of unknown cause, or a subject with infertilityattributed to an autoimmunologic factor (anti-phospholipid antibodysyndrome) is also suitable as a subject for administration of themedicine of the present invention (also see Examples). Particularly, (1)a subject with infertility that is presumed to be primarily caused byenhanced immunological mechanism of excluding a fertilized ovum or anembryo from the mother's body as a foreign material due to immunologicalabnormalities, and/or (2) a subject with infertility that is presumed tobe primarily caused by defective placenta construction due toimmunological abnormalities, is suitable as a subject for administrationof the medicine of the present invention.

As described above, sterility and infertility accompanied by autoimmunediseases other than anti-phospholipid antibody syndrome are also subjectfor administration of the medicine of the present invention. Examples ofsuch autoimmune diseases include systemic lupus erythematosus,ANCA-associated vasculitis (cryoglobulinemic vasculitis, IgA vasculitis,hypocomplementemic urticarial vasculitis, polyarteritis nodosa, Kawasakidisease, anti-GBM disease, Takayasu's arteritis, giant cell arteritis,microscopic polyangiitis, granulomatosis with polyangiitis, andeosinophilic granulomatosis with polyangiitis), and malignant rheumatoidarthritis. Autoimmune diseases including anti-phospholipid antibodysyndrome may cause defective construction of the placenta throughthrombus formation or the like. Defective construction of the placentamay also be accompanied by vasculitis of the placenta and inflammationof the placenta, in addition to thrombus formation. It can be expectedto suppress undesirable activity of autoantibodies and to amelioratedefective construction of the placenta, by administering the medicine ofthe present invention.

Defective Pregnancy Conditions Other Than Sterility and Infertility

The term “defective pregnancy conditions other than sterility andinfertility” according to the present specification includes illnessesor diseases occurring along with pregnancy (pregnancy complications andthe like) such as, for example, pregnancy-induced hypertension syndrome,thrombosis, infarction, cardiac failure (peripartum cardiomyopathy orthe like), and pulmonary edema; however, the term is a concept that alsoincludes conditions which are defective compared to the normal state ofpregnancy, in addition to conditions which are classified as illnessesor diseases. Among these, a subject with a high potential of the onsetof pregnancy-induced hypertension syndrome (for example, having an onsethistory of pregnancy-induced hypertension syndrome) is more suitable asthe subject for administration of the medicine of the present invention.Although the mechanism is not clearly known, since the immunologicalrelations between the mother and the child is improved by thistreatment, placenta construction proceeds smoothly. Also, since themother's body is also subjected to less stress, a possibility ofavoiding pregnancy complications can also be expected.

Pregnancy-induced hypertension syndrome refers to a syndrome whichdevelops hypertension, which is observed during pregnancy, mainly in thelatter half of pregnancy and until the 12^(th) week after delivery, ordevelops hypertension and symptoms such as edema and albuminuriaoccurring concomitantly with hypertension. The disease type ofpregnancy-induced hypertension syndrome is more specifically classifiedinto pregnancy-induced hypertension, preeclampsia, superimposedpreeclampsia, and eclampsia. Pregnancy-induced hypertension syndrome isrecurrent, and in the case of having a medical history ofpregnancy-induced hypertension syndrome during the previous pregnancy,there is a high risk of developing pregnancy-induced hypertensionsyndrome again during the subsequent pregnancy.

The causes of pregnancy-induced hypertension syndrome include vascularendothelial disorders caused by placental ischemia and a hypoxic state.Therefore, in the case of having pregnancy-induced hypertensionsyndrome, defective construction of the placenta may occur. Furthermore,as a result, there is a possibility that fetal growth restriction,abruption of the placenta, fetus malfunction, and fetal death may occur.

In the case of a patient of pregnancy-induced hypertension syndrome, itcan be expected that rejection against trophoblast, which is a fetalcomponent, is suppressed (suppression of a kind of immunologicalabnormalities) by administering the medicine of the present invention tothe patient, and reconstruction of endometrial spiral arteries intolarger spiral arteries is promoted. Therefore, satisfactory constructionof the placenta can be promoted.

Age of Subject for Administration of Medicine

The age of the subject for administration may be any age capable ofchildbearing. When the subject for administration is a human being, forexample, the age is preferably from age 16 to age 50, more preferablyfrom age 16 to age 45, and even more preferably from age 16 to age 40.If the age is 16 or higher, consent of the person herself may beobtained. Furthermore, in the case of a sterile patient, since thepossibility of secure establishment of pregnancy is higher if the age ofthe patient is 40 or lower, the effects of the medicine according to thepresent invention can be obtained more efficiently. The age of thesubject for administration is not limited to this; however, for example,the age is from age 30 to age 45, from age 33 to age 43, from age 33 toage 41, or from age 33 to age 39.

More Preferred Examples of Subject for Administration of Medicine

It is preferable that the subject for administration of the medicinedescribed above does not have any disease, illness or condition otherthan the above-mentioned defective pregnancy conditions, andparticularly, it is more preferable that the subject for administrationdoes not have any disease, illness or abnormality related to the uterus,such as submucosal myoma of the uterus, endometrial polyps, intrauterineadhesion, congenital abnormalities in the uterus, and hydrosalpinx.Furthermore, it is preferable that the subject for administration doesnot have any disease, illness or condition with a high risk of beingseriously affected by the medicine of the present invention.

Subject for Administration of Medicine and Th1/Th2 Cell Ratio

According to an embodiment, the Th1/Th2 cell ratio in the subject foradministration of the medicine may be determined, and this ratio may beused for the determination of the necessity of the administration of themedicine to a relevant subject or the detei iination of the dose of themedicine (see Examples for the details of this process). For example,the Th1/Th2 cell ratio in a subject for administration of the medicineis at an increased level compared to a normal person. In a more specificexample, a subject whose Th1/Th2 cell ratio has increased by 10% to 800%compared to a normal person is a preferred subject for administration ofthe medicine of the present invention; a subject whose Th1/Th2 cellratio has increased by 10% to 400% is a more preferred subject foradministration of the medicine of the present invention; and a subjectwhose Th1/Th2 cell ratio has increased by 10% to 200% may be an evenmore preferred subject for administration of the medicine of the presentinvention. Regarding specific values of the Th1/Th2 cell ratio, asubject having a Th1/Th2 cell ratio of 10 to 50 is a preferred subjectfor administration of the medicine of the present invention, a subjecthaving a Th1/Th2 cell ratio of 10 to 40 is a more preferred subject foradministration of the medicine of the present invention, and a subjecthaving a Th1/Th2 cell ratio of 10 to 30 may be an even more preferredsubject for administration of the medicine of the present invention. Inregard to the Examples that will be described below, the average valueof the Th1/Th2 cell ratio of normal persons was about 7 to 8, and themaximum value of the Th1/Th2 cell ratio in a treated group was about 30to 40.

Here, a normal person refers to an individual who does not havedefective pregnancy conditions that serves as a target of amelioration.For example, in a case in which amelioration of sterility is intended, anormal person refers to an individual who does not suffer at least fromsterility, and in a case in which amelioration of infertility isintended, a noiiiial person refers to an individual who does not sufferat least from infertility. Preferably, a normal person refers to anindividual who does not have defective pregnancy conditions that serveas a target of amelioration, and has not developed any other existingillness or disease (however, may have defective pregnancy conditionsother than those conditions serving as targets of amelioration). Morepreferably, a normal person refers to an individual who does not haveall the above-mentioned defective pregnancy conditions, and even morepreferably, a normal person refers to an individual who does not haveall the above-mentioned defective pregnancy conditions and has notdeveloped any other existing illness or disease.

Without being particularly limited, it is preferable that determinationof the Th1/Th2 cell ratio is carried out at a time point between anytime immediately before the scheduled day of performing administrationof the medicine (the scheduled day, the day before, or two before) andany time after 6 months; a time point between any time immediatelybefore the scheduled day of performing administration of the medicineand any time after 3 months is more preferred; a time point between anytime immediately before the scheduled day of performing administrationof the medicine and any time after 2 months is even more preferred; anda time point between any time immediately before the scheduled day ofperforming administration of the medicine and any time after 1 month isparticularly preferred. In a more specific example, in a case in whichthe subject for administration of the medicine is receiving a treatmentsuch as IVF-ET or ICSI, it is preferable to perfoni determination of theTh1/Th2 cell ratio during the menstrual cycle immediately before, twocycles before, three cycles before, four cycles before, five cyclesbefore, or six cycles before the day of receiving treatment; it is morepreferable to perform determination of the Th1/Th2 cell ratio during themenstrual cycle immediately before or two cycles before the day ofreceiving treatment; and it is even more preferable to performdetermination of the Th1/Th2 cell ratio during the menstrual cycleimmediately before the day of receiving treatment. When the effectexerted by the menstrual cycle on the immune state is considered, it ispreferable that the determination of the Th1/Th2 cell ratio is perfonled at relatively the same time point during the menstrual cycle in anormal person and a subject for administration of the medicine. It isalso preferable that the determination of the Th1/Th2 cell ratio isperformed during the low body temperature period (from the menstrualperiod to ovulation) of the menstrual cycle.

Without being particularly limited, more specifically, for example, thedetermination of the Th1/Th2 cell ratio in a subject with sterility canbe carried out before the scheduled day of performing administration ofthe medicine, preferably at least during the period until 1 or 2 daysbefore to 180 days before, more preferably during the period until 1 or2 days before to 60 days before, and even more preferably during theperiod until 2 days before to 30 days before, the implantation of afertilized ovum or a transplanted embryo. Without being particularlylimited, more specifically, for example, the determination of theTh1/Th2 cell ratio in a subject who can have defective pregnancyconditions other than sterility (infertility and the like) can becarried out before the scheduled day of performing administration of themedicine, preferably during the period of from 0 days to 60 days, morepreferably during the period of from 0 days to 30 days, even morepreferably during the period of from 0 days to 20 days or from 0 days to15 days, and particularly preferably during the period of from 0 days to10 days, after confirmation of pregnancy.

The Th1/Th2 cell ratio may be the value of any tissue in the body of thesubject for administration of the medicine; however, the ratio ispreferably the value of peripheral blood.

Particularly Specific Examples of Subject for Administration of MedicineInfecundity

A particularly preferred example of patient is a patient who meets thefollowing conditions (1) and (2), more preferably a patient who alsomeets the condition (3), and even more preferably a patient who alsomeets the conditions (4) to (6).

(1) A patient of implantation disorder, in whom, among examples ofsevere infecundity without clearly acknowledged causes, even if embryotransplantation of a fertilized ovum (regardless of being a freshembryo, a frozen embryo, an early stage embryo, or a blastocyst) havinga clearly recognizable satisfactory shape, is carried out 4 or moretimes, scientific pregnancy (increased hCG) is not achieved.

(2) A patient at the age of below 40. (3) A patient having a high valueof Th1/Th2 cell ratio, who is found when subjects are subjected to animmunological examination (screening) during the low body temperatureperiod (from the menstrual period to ovulation) with satisfactory bodycondition before pregnancy.

(4) A patient who does not have any active infectious disease.

(5) A patient who does not have any sustained infectious disease of HBV,HCV, or HIV.

(6) A patient who does not have a medical history of hypersensitivityagainst tacrolimus component.

Infertilitas

A particularly preferred example of patient is a patient who meets thefollowing conditions (1) and (2) (patients of infertilitas of unknowncauses are included therein), and more preferably a patient who alsomeets the conditions (3) to (5).

(1) A patient of severe infertilitas in whom clear abnormal findingsrelated to infertility are not recognized from a serological test.

(2) A patient of habitual abortion resistant to treatment (includinganti-phosphorus antibody syndrome).

(3) A patient who does not have any active infectious disease.

(4) A patient who does not have any sustained infectious disease of HBV,HCV, or HIV.

(5) A patient who does not have a medical history of hypersensitivityagainst tacrolimus component.

General Terms

Without being particularly limited, patients who continuously receiveadministration of tacrolimus or a derivative thereof for a medicalpurpose other than amelioration of pregnancy conditions (for example, apatient who is continuously administered with tacrolimus after organtransplantation) may be excluded from the subject for administration ofthe medicine of the present invention.

Route of Administration/Method for Administration

The method for administration (route of administration) of the medicineof the present invention can be appropriately determined based on theage and condition of the subject for administration, the duration oftreatment, and the like. Specifically, both oral administration andparenteral administration may be employed; however, oral administrationis preferred (oral administration is employed in Examples). Examples ofparenteral administration include methods such as administration byinjection, administration using a suppository, and administration usinga percutaneous absorption type preparation. Examples of the type ofadministration by injection include intramuscular injection,intraperitoneal injection, subcutaneous injection, intravenousinjection, and local injection. Furthermore, the medicine of the presentinvention can be administered through various routes such aspercutaneous, transnasal, transvaginal, and transrectal routes.

Dosage

The dosage of the medicine varies depending on the type of the disease,illness or condition of the patient who receives administration of themedicine, severity, results of various examinations, the type of theactive ingredient of the medicine, and the like. Furthermore, the dosageof the medicine also varies depending on the age of the patient to betreated, the number of times of treatment according to the treatmentmethod of the present invention, results of various examinations, andthe like. For instance, from the viewpoint of the content of the activeingredient included in the medicine, the medicine of the presentinvention is administered at a dose that is lower than the dosage in thecase in which the medicine is used as an immunosuppressant in thetreatment for living donor organ transplantation and immune systemdiseases. For example, in a case in which the subject for administrationof the medicine is a human being, without being particularly limited,the medicine is administered in an amount, as an amount of the activeingredient, preferably in the range of 0.5 to 5 mg or 1 to 5 mg, morepreferably in the range of 0.5 to 4.5 mg or 1 to 4.5 mg, even morepreferably in the range of 0.5 to 4 mg or 1 to 4 mg, still morepreferably in the range of 0.5 to 3.5 mg or 1 to 3.5 mg, even morepreferably in the range of 0.5 to 3 mg or 1 to 3 mg, and most preferablyin the range of 1 to 2 mg, per day. Hereinafter, unless particularlystated otherwise, the description concerning the dosage of the medicineis applicable in the case in which the subject is a human being, and thedosage is disclosed as the amount of the active ingredient.

Furthermore, without being particularly limited, in the case of oraladministration, the frequency of administration per day is preferably 1to 4 times, more preferably 1 to 3 times, and even more preferably 1 to2 times.

If necessary, the Th1/Th2 cell ratio described above (also see Examples)may be determined in advance, and the dosage may be determined based thevalue of the ratio. In regard to continuous administration for a certainperiod of during pregnancy and after delivery, including the treatmentfor infertility, the Th1/Th2 cell ratio may be measured at a constantinterval during the duration of treatment, and the dosage may bedetermined based on the measurement results every time the cell ratio ismeasured anew.

As an example of determining the dosage based on the value of theTh1/Th2 cell ratio, in a case in which the Th1/Th2 cell ratio is 10 orhigher, the medicine is administered in an amount, as an amount of theactive ingredient, preferably in the range of 0.5 to 5 mg or 1 to 5 mg,more preferably in the range of 0.5 to 4.5 mg or 1 to 4.5 mg, even morepreferably in the range of 0.5 to 4 mg to 1 to 4 mg, still morepreferably in the range of 0.5 to 3.5 mg or 1 to 3.5 mg, even morepreferably in the range of 0.5 to 3 mg or 1 to 3 mg, and most preferablyin the range of 1 to 2 mg, per day. As another example, in a case inwhich the Th1/Th2 cell ratio is in the range of from 10 to 13, themedicine is administered in an amount, as an amount of the activeingredient, preferably in the range of 0.5 to 3 mg or 1 to 3 mg, morepreferably in the range of 0.5 to 2 mg to 1 to 2 mg, and even morepreferably in the range of 0.5 to 1 mg, per day. As another example, ina case in which the Th1/Th2 cell ratio is in the range of higher than 13and 16 or lower (for example, 15.8 or less), the medicine isadministered in an amount, as an amount of the active ingredient,preferably in the range of 0.5 to 4 mg or 1 to 4 mg, more preferably 0.5to 3.5 mg or 1 to 3.5 mg, and even more preferably in the range of 1 to3 mg or 1.5 to 2.5 mg, per day. As another example, in a case in whichthe Th1/Th2 cell ratio is 15 or higher (for example, in the case ofbeing higher than 15.8), the medicine is administered in an amount, asan amount of the active ingredient, preferably in the range of 0.5 to 5mg or 1 to 5 mg, more preferably in the range of 0.5 to 3.5 mg or 1 to3.5 mg, and even more preferably in the range of 2 to 3.5 mg or 2.5 to3.5 mg, per day.

The dosage of the medicine can be increased in accordance with thenumber of times of performing the amelioration method according to thepresent invention (number of times of pregnancy). For example, everytime the number of times of treatments is traced during the treatmentfor sterility (an embodiment of amelioration), the dose per day isincreased preferably by 0.5 to 3 mg (amount of the active ingredient;hereinafter, the same), more preferably by 0.5 to 2 mg, and even morepreferably by 0.5 to 1 mg, from the dose at the time of treatment of theprevious time. That is, with regard to the dose per day of the medicinethat is administered from the beginning to the end of the first durationof treatment, the dose per day for the second duration of treatment thatis carried out subsequently to the first duration of treatment, isincreased by 0.5 to 3 mg. This is repeated every time for a singleduration of treatment until pregnancy is established.

Regarding the number of times of treatments for the treatment ofsterility (an embodiment of amelioration), preferably, repetition of 2times to 5 times is tolerated, and more preferably, repetition of 2times to 4 times is tolerated. In regard to the repetition of treatment,it is preferable that the dose per day at the end of the final durationof treatment does not exceed the dose that is preferable for thetreatment method of the present invention (up to 5 mg as the amount ofthe active ingredient, per day).

Furthermore, in regard to the treatment of infertility (an embodiment ofamelioration), during the gestation period, the Th1/Th2 cell ratio maybe measured at a constant interval, and every time the Th1/Th2 cellratio has increased, the dose per day may be increased preferably by 0.5to 3 mg (amount of the active ingredient; hereinafter, the same), morepreferably by 0.5 to 2 mg, and even more preferably by 0.5 to 1 mg,every time.

The dosage described above provides values that are applicable to womenin general; however, when a woman has a body weight that is extremelyfar from the standard body weight (for example, a woman having a bodyweight of less than 45 kg or a woman having a body weight of more than75 kg) is the subject, the dosage per kg of body weight may bedetermined by dividing the dosage by 60 (kg), and thereby the actualdosage may be adjusted according to the actual body weight of thesubject.

In the case of using a medicine including rapamycin or a derivativethereof as the active ingredient, an appropriate dosage in accordancewith the type of the active ingredient can be set; however, in additionto the embodiment of applying the above-mentioned dosage as an example,an embodiment of applying a dosage obtained by multiplying theabove-mentioned dosage by ⅔ may also be employed. In the case of using amedicine including cyclosporin or a derivative thereof as the activeingredient, an appropriate dosage in accordance with the type of theactive ingredient can be set; however, for example, an embodiment ofapplying a dose of 1,000 mg or less, and preferably 60 mg or less, perday as the amount of the active ingredient may also be employed.

Timing and Duration of Administration of Medicine

The timing and duration of the administration of the medicine of thepresent invention vary depending on the disease, illness or condition asan object of amelioration. In a case in which a human subject suffersfrom sterility, it is preferable that the medicine is administered atleast for a period from preferably 1 or 2 days before to 60 days before,more preferably 2 to 30 days before, or even more preferably 2 to 15days before the implantation of a fertilized ovum or a transplantedembryo into the endometrium, until preferably 0 to 100 days after, ormore preferably 0 to 15 days after (0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 days after) the implantation. When the subject isa patient who has received a treatment of artificial insemination or invitro fertilization, the administration of the medicine is carried outfor a period from preferably 1 or 2 days to 60 days before, morepreferably 2 to 30 days before, and even more preferably 2 to 15 daysbefore sperm injection or embryo transplantation until preferably 0 to100 days after, and more preferably 0 to 15 days after (0 to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days after) sperm injection orembryo transplantation.

Furthermore, in a case in which a human subject suffers from infertility(pregnancy loss), it is preferable that the medicine is continuouslyadministered preferably from the day of confirming establishment ofpregnancy until after 200 days, more preferably from the day ofconfirming establishment of pregnancy until after 300 days, or even morepreferably from the day of confirming establishment of pregnancy untilthe delivery.

In a case in which a human subject has a risk of having pregnancycomplications (including pregnancy-induced hypertension syndrome), it ispreferable that the medicine is continuously administered preferablyfrom the day of confirming establishment of pregnancy until after 200days, more preferably from the day of confirming establishment ofpregnancy until after 300 days, even more preferably from the day ofconfirming establishment of pregnancy until the delivery, or mostpreferably from the day of confirming establishment of pregnancy to 60days after the delivery.

Furthermore, the dose can also be appropriately modified at any stage ofthe first trimester, the second trimester, or the third trimester ofpregnancy. For example, as the subject goes through each stage of thefirst trimester, the second trimester, and the third trimester ofpregnancy and the gestation period described below, the dose of themedicine per day may be reduced stepwise by 0.5 to 1 mg. Alternatively,an embodiment of administering the medicine only during the firsttrimester of pregnancy and stopping administration from the secondtrimester may be employed; an embodiment of continuing administrationuntil the second trimester of pregnancy but stopping administration fromthe third trimester may be employed; or an embodiment of administeringthe medicine only during the first trimester and the third trimester ofpregnancy and pausing administration during the second trimester mayalso be employed.

Furthermore, the duration of continuous administration may also beregulated depending on whether the examination results for pregnancyconditions at each stage of pregnancy are satisfactory. From 100 daysafter the establishment of pregnancy, the mother's body enters a stableperiod of pregnancy. Therefore, the dose of the medicine is reduced asfar as possible so long as sufficient therapeutic effects are obtained,and thereby the influence of adverse effects of the medicine on themother's body and the fetus is suppressed to the minimum. Thus, thephysical and burden on the subject for administration can be reduced tothe minimum.

2. Method for Ameliorating Pregnancy Conditions

A method for ameliorating pregnancy conditions using the medicineaccording to the present invention (an embodiment thereof is a methodfor treating defective pregnancy conditions) is also within the scope ofthe present invention. The details of the method for amelioratingpregnancy conditions of the present invention are hereinafter explained.

The method for ameliorating pregnancy conditions according to anembodiment of the present invention includes a step of administering themedicine according to the present invention to a subject (administrationstep). The term amelioration of pregnancy conditions means thatdefective pregnancy conditions are ameliorated to a satisfactory stateor a normal state, and this is a concept that also includes preventionof acquiring defective pregnancy conditions. Specific examples of thedefective pregnancy conditions include sterility, infertility, andpregnancy complications such as pregnancy-induced hypertension syndrome.The disease, illness, and condition included in the concept of defectivepregnancy conditions are as explained in the aforementioned section of(Subject for administration of medicine) in [1. Medicine (pharmaceuticalcomposition)]. Regarding the active ingredient, the medicine to beadministered contains a compound represented by General Formula (I)described above or a pharmaceutically acceptable salt thereof as anactive ingredient, or contains cyclosporin, rapamycin, or a derivativethereof (including a pharmaceutically acceptable salt thereof) as activeingredients. Also in regard to the medicine according to the presentinvention, the matters explained in the aforementioned section [1.Medicine (pharmaceutical composition)] are included. The subject towhich the therapeutic method of the present invention is applied is asexplained in the section of (Subject for administration of medicine) in[1. Medicine (pharmaceutical composition)].

The therapeutic method of the present invention according to anotherembodiment may further include, in addition to the administration step,(1) a step of measuring the Th1/Th2 cell ratio, and/or (2) a step ofperfoi ling artificial insemination (injection of sperms into the womb)or transplanting a fertilized ovum or an embryo obtained by in vitrofertilization into the endometrium. The various steps are explained inmore detail below.

Administration Step

The administration step is a step of administering the medicineaccording to the present invention to a subject.

When the method of the present invention is a method for ameliorating(including a therapeutic method) sterility in a human subject, regardingthe period for administration of the medicine in the administrationstep, it is preferable that the medicine is administered at least for aperiod from preferably 1 or 2 days to 60 days before, more preferably 2to 30 days before, or even more preferably 2 to 15 days before theimplantation of a fertilized ovum or a transplanted embryo into theendometrium until preferably 0 to 100 days after, or more preferably 0to 15 days after, the implantation. When the human subject is a patientwho has received treatment for artificial insemination or in vitrofertilization, administration is carried out for a period frompreferably 1 or 2 days to 60 days before, more preferably 2 to 30 daysbefore, or even more preferably 2 to 15 days before sperm injection orembryo transplantation, and until preferably 0 to 100 days after, ormore preferably 0 to 15 days after spec injection or embryotransplantation.

When the method of the present invention is a method for ameliorating(including a therapeutic method) infertility in a human subject, theperiod for administration of the medicine in the administration step ispreferably from the day of confirming establishment of pregnancy untilafter 200 days, more preferably from the day of confirming establishmentof pregnancy until after 300 days, or even more preferably from the dayof confirming establishment of pregnancy until the delivery.

When the method of the present invention is a method for ameliorating(also including prevention of) pregnancy complications such aspregnancy-induced hypertension syndrome in a human subject, the periodfor administration of the medicine in the administration step ispreferably from the day of confirming establishment of pregnancy untilafter 200 days, more preferably from the day of confirming establishmentof pregnancy until after 300 days, even more preferably from the day ofconfirming establishment of pregnancy until the delivery, or mostpreferably from the day of confirming establishment of pregnancy until60 days after the delivery.

Administration during the above-mentioned period may be continuous (thatis, administered everyday during the period), or may be intermittent(that is, days without administration may be included during theperiod). More particularly, the description in (Timing and duration ofadministration of medicine) of [1. Medicine (pharmaceuticalcomposition)] is applied. Specific examples of intermittentadministration include an embodiment of administering the medicine withregularity of once in predetermined dates (1 day, 2 days, or 3 days),and an embodiment of administering the medicine one or morepredetermined stoppage period(s) composed of a plurality of days.

The dosage is as described in the section of (Dosage) in [1. Medicine(pharmaceutical composition)].

Step of Measuring Th1/Th2 Cell Ratio

Furtheunore, the method according to another embodiment of the presentinvention may further include a step of measuring the Th1/Th2 cellratio. The Th1/Th2 cell ratio thus determined may also be used for thedetennination of the necessity of administration of the medicine to thesubject, or the determination of the dosage of the medicine, asdescribed in the section [1. Medicine (pharmaceutical composition)]. Thestep of measuring the Th1/Th2 cell ratio is carried out before theadministration step. Here, the term “before the administration step”refers to the time between an administration and another administrationduring a single period of administration, and also refers to a timebefore the initial administration during a single period ofadministration (treatment period).

When the method of the present invention is a method for amelioratingsterility, the step of measuring the Th1/Th2 cell ratio can be carriedout, for example, before the scheduled day of performing administrationof the medicine, preferably during a period of from 1 or 2 days to 180days before, more preferably a period of from 1 or 2 days to 60 daysbefore, and even more preferably a period of from 2 days to 30 daysbefore the implantation of a fertilized ovum or a transplanted embryo.

When the therapeutic method of the present invention is a therapeuticmethod for ameliorating defective pregnancy conditions other thansterility, including infertility and pregnancy complications such aspregnancy-induced hypertension syndrome, the step of measuring theTh1/Th2 cell ratio can be carried out before the scheduled day ofperforming administration of the medicine, preferably during a period offrom 0 days to 60 days, more preferably a period of from 0 days to 30days, even more preferably a period of from 0 days to 20 days or from 0days to 15 days, or particularly preferably a period of from 0 days to10 days after the confirmation of pregnancy.

Step of Performing Artificial Insemination or Transplanting FertilizedOvum or Embryo Obtained by in Vitro Fertilization Into Endometrium

Upon applying the method for ameliorating pregnancy conditions using themedicine according to the present invention, the subject (including ahuman subject) may receive a treatment of artificial insemination(injection of sperms into the womb), or may receive a treatment oftransplanting a fertilized ovum or an embryo obtained by in vitrofertilization into the endometrium. Ameliorating the pregnancyconditions related to a fertilized ovum or a transplanted embryo thathas been implanted after receiving a the said treatment is aparticularly preferred embodiment of the present invention. Specificmethods for in vitro fertilization include methods such as IVF-ET andICSI. IVF-ET is further classified into fresh embryo transplantation(fresh ET) of using a fresh embryo for transplantation and freeze-thawembryo transplantation (FET) of using a frozen and thawed embryo thathas been stored in a frozen state after collectiono for transplantation.Among them, FET is preferable. When the medicine of the presentinvention is administered, the efficiency of establishment of pregnancyand childbirth of a subject who has received these therapeutic methodscan be increased, and the onset of pregnancy complications, etc. canalso be suppressed.

However, the establishment of pregnancy in a subject with sterility orinfertility may be based on spontaneous impregnation, and amelioratingthe pregnancy conditions in spontaneous impregnation is also included inthe scope of the present invention.

4. Other Embodiments

Furthermore, use of at least one compound selected from Compound (I)described above, rapamycin or a derivative thereof, cyclosporin or aderivative thereof, and pharmaceutically acceptable salts thereof, forthe production of a medicine for ameliorating pregnancy states is alsoincluded in the scope of the present invention.

5. Summary

That is, the scope of the present invention includes the followingembodiments.

1) A medicine for ameliorating pregnancy conditions, containing acompound represented by General Formula (I) described above (Compound(I)) or a pharmaceutically acceptable salt thereof as an activeingredient.

2) The medicine according to 1), wherein the compound is tacrolimus or apharmaceutically acceptable salt thereof.

3) The medicine according to 1) or 2), which ameliorates defectivepregnancy conditions attributed to immunological abnormalities.

4) The medicine according to any one of 1) to 3), which ameliorates atleast one type of defective pregnancy condition selected from the groupconsisting of sterility, infertility, and pregnancy-induced hypertensionsyndrome.

5) The medicine according to 4), wherein the aforementioned sterilityand infertility are accompanied by an autoimmune disease.

6) The medicine according to 5), wherein the autoimmune disease isanti-phospholipid antibody syndrome.

7) The medicine according to any one of 1) to 6), wherein the medicineis administered to a subject of treatment with an amount of 5 mg or lessper day of the aforementioned active ingredient.

8) The medicine according to 7), wherein the amount of theaforementioned active ingredient is in the range of from 1 mg to 3 mgper day.

9) The medicine according to any one of 1) to 8), wherein the medicineis administered to a subject who has an increased Th1/Th2 cell ratiocompared to a healthy person.

10) The medicine according to any one of 1) to 9), wherein the medicineis administered to a subject who has received to transplantation of anembryo obtained by in vitro fertilization.

11) The medicine according to any one of 1) to 9), wherein the medicineis administered at least during a period from 1 or 2 days before theimplantation of a fertilized ovum or a transplanted embryo into theendometrium, to 0 days after the implantation in a case in which themedicine is intended for sterility as a defective pregnancy condition;and the medicine is administered at least for a period from theestablishment to the 200^(th) day of pregnancy in a case in which themedicine is intended for a defective pregnancy condition other thansterility.

12) A method for ameliorating a pregnancy condition, including a step ofadministering a medicine containing aforementioned compound (I) or apharmaceutically acceptable salt thereof as an active ingredient forameliorating pregnancy conditions, wherein the medicine includes.

13) The method according to 12), wherein the aforementioned medicine isadministered by any one form or embodiment according to 2) to 11).

Example 1

-   Objective

We, the inventors, evaluated the clinical efficacy of immunosuppressivetreatment with tacrolimus for repeated implantation failure (RIF)patients who have elevated in T helper (Th) 1/Th2 cytokine producingcell ratios (Th1/Th2 cell ratio).

-   Method of Study:

This was a prospective cohort study of treatment for RIF patients (n=42)with elevated peripheral blood Th1 (CD4⁺/IFN-γ⁺)/Th2 (CD4⁺/IL-4⁺) cellratios at the Sugiyama clinic between November 2011 and October 2013.Twenty-five patients were treated with tacrolimus (treatment group) and17 received no treatment (control group).

Treatment group continuously received tacrolimus from two days beforeembryo transfer until the day of the pregnancy test, for a total of 16days. The daily dose of tacrolimus (1-3 mg) was determined based on thedegree of the Th1/Th2 cell ratio.

-   Results:

The clinical pregnancy rate of the treatment group was 64.0%, which wassignificantly higher than that of the control group (0%) (P<0.0001). Inthe treatment group, the miscarriage rate was 6.3%, the live birth ratewas 60.0% (P<0.0001). There was no significant side effect fromtacrolimus in treatment group. No one developed obstetricalcomplications during pregnancy.

-   Conclusion:

An immunosuppressive treatment using tacrolimus improved pregnancyoutcome of repeated implantation failure patients with elevated Th1/Th2cell ratios.

INTRODUCTION

Incidence of in-vitro fertilization (IVF) and embryo transfer (ET) hasbeen soared recent years world-wide.¹ This was accompanied by anincrease in number of women with multiple IVF failures, includingrepeated implantation failures (RIF). When conducting IVF/ET, an embryois transferred to the uterine cavity between 2 to 5 days afterfertilization. Pregnancy is established when an embryo, which is so tospeak a semi-allograft, is successfully implanted to maternal deciduawith an establishment of maternal immune tolerance.² The establishmentof proper immune responses at the time of implantation is a key forsuccessful implantation. Hence, immune etiology may play a key role inRIF after IVF/ET.

T helper (Th) 1 and Th2 cells play important roles in immune responses,such as immune rejection or tolerance.³ There is a general agreementthat pregnancy is associated with Th2 dominance, and Th1 immune responseis associated with embryonic rejection.^(4,5) An underling mechanism ofembryo rejection is considered to be similar to an allograft rejection.⁶A transferred embryo during IVF/ET may fail to implant due to thesimilar immunological reaction involved in allograft rejection.

In the past decades the graft survival rate has been dramaticallyimproved.⁷ This success can be attributed to the development of newimmunosuppressive agents. Tacrolimus (Prograf®, Astellas Pharma, Tokyo)is one of the major immune-suppressive agents that have been utilizedafter allogeneic organ transplantation to reduce the allo reactivity ofa recipient's immune system, thereby lowering the risk of organrejection.⁸ Tacrolimus has been reported to suppress the immunologicalrejection of an allo-graft and promote its survival by inhibitinglymphocyte proliferation induced by allo-antigens, cytotoxic T cellgeneration, expression of IL-2 receptor and the production of T cellderived soluble mediators such as IL-2 and IFN-γ.⁹ Tacrolimus has beenreported to effectively control graft versus host disease or otherimmunological disorder such as rheumatoid arthritis,^(10, 11) althoughdifferent subsets of T-lymphocytes are associated with these conditions.Women with RIF have increased Th1 immune responses with increasedperipheral blood Th1/Th2 cell ratio.⁴ Therefore, an immune-suppressiveagent, such as tacrolimus might improve implantation rates and pregnancyoutcome in women who have a history of RIF after ART cycles,particularly with increased Th1 immunity response. In this study, weinvestigated the clinical efficacy of tacrolimus for women with RIF andan elevated peripheral blood Th1/Th2 cell ratio.

Materials and Methods

-   Study Population

A total of 81 patients with a history of 5 or more RIF after IVF/ETcycles were consecutively enrolled in the study at the division ofReproductive Medicine, the Sugiyama clinic, Tokyo, Japan, betweenNovember 2011 and February 2014. This study was approved by theInstitutional Review Board of the Sugiyama Clinic. A signed informedconsent form was obtained from all patients prior to entering the study.

Study patients were selected if they were eligible for IVF/ET, and had ahistory of 5 or more failed IVF/ET cycles with morphologically anddevelopmentally good-quality embryos into an adequately preparedendometrium (endometrial thickness ≤8 mm). Study patients were assessedby transvaginal ultrasound, hysterosalpingography, and hysteroscopybefore the index ART cycle. None of the participants had submucosalfibroids, endometrial polyps, intrauterine adhesions, congenitalanomalies of uterus or hydrosalpinges. In addition, none of the womenhad any history of autoimmune disease. Pregnant women or women withchronic medical or inflammatory conditions were excluded. Women whomiscarried in a former IVF cycle or had IVF cycles, or receivedvaccination in 3 months were excluded in the study. Women with acquiredor inherited thrombophilia were also excluded in this study.

Blood was drawn between cycle day (CD) 5 and 10 prior to the index ARTcycle for the evaluation of a baseline value of Th1/Th2 cell ratios.Forty-two patients had elevated Th1/Th2 cell ratio (≥10.3) and 39 hadnormal Th1/Th2 cell ratio (<10.3). Normal ranges for Th1/Th2 cell ratioswere established using 28 women who had a history of normal delivery byeither natural conception or artificial insemination with husband sperm.Blood was drawn between CD5 and CD10 of the menstrual cycle. A Th1/Th2cell ratio equal to 10.3 or above was classified as an elevated Th1/Th2cell ratio, which was determined by the mean plus one standard deviationof Th1/Th2 cell ratio levels.

-   Analyses of the Th1 and Th2 Cells

For the evaluation of a baseline value of Th1/Th2 cell ratios, total 10ml of venous blood was obtained. Th1 and Th2 cells were determined bydetecting the intracellular interferon (IFN)-γ and IL-4 production.

The specific staining of lymphocytes was performed by incubating wholeblood with anti-CD4-PC5 or anti-CD8-PC5-conjugated monoclonal antibodies(mAbs) (Beckman Coulter, Fullerton, Calif., USA). The red blood cells(RBCS) were then removed by lysis (FACS Lysing solution; BectonDickinson, BD Biosciences, Franklin Lake. N.J., USA) and the lymphocyteswere analyzed using flow cytometry (FACSCalibur; Becton Dickson). Aftersurface staining of the activated whole blood samples with anti-CD4-PC5conjugated mAbs, RBC lysis and specific intracellular staining usingFastImmune™ IFN-γ-FITC/IL-4-PE (Becton Dickinson) were subsequentlyperformed according to the manufacturer's instructions. Th1 cells weredefined as CD4⁺ lymphocytes with intracellular IFN-γ but without IL-4.Th2 cells were detected as CD4⁺ lymphocytes with intracellular IL-4 butwithout IFN-γ. The ratio of IFN-γ to IL-4 positive Th cells wasexpressed as the Th1/Th2 cell ratio.

-   Tacrolimus (Prograf®) Treatment

In 42 patients with elevated Th1/Th2 cytokine ratios (equal or above10.3), 25 of them were treated with tacrolimus (Prograf®, AstellasPharma, Tokyo; treatment group), and the others (n=17) were not treated(control group) during the index IVF/ET cycles. The patients in thetreatment group began tacrolimus two days prior to ET and continueduntil the day of pregnancy test, for a total of 16 days. The dailydosage of tacrolimus was 1 to 3 mg depending on the degree of Th1/Th2cell ratio elevation; patients (n=12) with mildly increased Th1/Th2 cellratio (≥10.3 and <13.0) were treated with 1 mg of tacrolimus, daily.Patients (n=8) with moderately increased Th1/Th2 cell ratio (≥13.0 and<15.8) were treated with 2 mg of tacrolimus daily. Patients (n=5) withhighly increased Th1/Th2 cell ratio (≥15.8) were treated with 3 mg oftacrolimus daily.

-   IVF-ET treatment

Ovarian stimulation and the oocyte pick-up (OPU) were performed as usualaccording to our previous report.¹² Our mild stimulation protocol was asfollows: patients took 50 mg of clomiphene citrate (Serophen®, MerckSerono, Tokyo) per day for 5 days between days 3 and 7 of the menstrualcycle, and 225 International Units (IU) of rec-FSH (Gonal-F®, MerkSerono, Tokyo,) were administered on days 4, 6 and 8 of the menstrualcycle. On day 10, when the dominant follicles reached ≥17 mm indiameter, either 10,000 IU of human chorionic gonadotropin (hCG;Gonadotropin, Mochida, Tokyo) was injected, or 300 μg of buserelinacetate (Buserecur, Fuji Pharma, Tokyo) was administered nasally, andOPU was performed 35 hours later. Additional rec-FSH (150 IU per a day)was administered as needed, based on follicular growth. Eitherintracytoplasmic sperm injection (ICSI) or conventional co-culture wasused depending on the semen parameters.

Embryos were placed to the patient's uterus transcervically using a softcatheter (Kitazato ET catheter, Kitazato Supply, Shizuoka, Japan). Inall patients, either one or two embryos were transferred. Fresh ET wasperformed on day 3. Any remaining embryos were cryopreserved usingvitrification.¹³ On the day of ET and 5 days after ET, 125 mg ofhydroxyprogesterone caproate (Progestone Depot-S, Fuji Pharma, Tokyo)was injected for luteal support. Chlonnadinone acetate (Lutoral®,Shionogi, Osaka) was administered orally for 14 days after ET. For thefrozen-thawed ET (FET) cycle, endometrial preparation was used eithernatural ovulatory cycle or hormone replacement cycle. Using naturalovulatory cycle the day of ET was defined as 3 days after the ovulation.The same luteal support as in the fresh ET cycle was administered. Usinghormonal replacement cycle (HRC), the uterine endometrium was preparedfor ET using conjugated estrogens (Premarin 0.625 mg, Wyeth, Tokyo,Japan) and transdermal estradiol (Estrana TAPE 0.72 mg, HisamitsuPharmaceutical, Tokyo, Japan). These treatments were administered fromthe 3rd day of the menstrual cycle or the 1st day without bleeding untilthe day of the urinary pregnancy test. Administration of progesterone(100 mg in oil; Progestone Depot-S, Fuji Pharmaceutical, Tokyo, Japan)was initiated on the 12th day of the menstrual cycle. Three days afterthe initiation of progesterone treatment, embryos were thawed and thosethat had survived were transferred.¹⁴

All patients received a transfer of one or two morphologicallygood-quality embryos (MGEs) 3 days after oocyte retrieval. For frozenembryo cycle, 3 day old frozen-thawed embryos were transferred 3 daysafter ovulation in natural ovulatory cycle or 3 days after progesteroneadministration in HRC. MGEs were defined as having 7 or more blastomeresand possessing less than 10% of fragmentation 3 days after oocyteretrieval.¹³ Pregnancy test was done 14 days after ET. A clinicalpregnancy was recognized when the development of a gestational sac wasdetected by transvaginal ultrasound 21 days after ET. An ongoingpregnancy was recognized when a normal fetus was seen by transvaginalultrasound at 12th week of gestation. The primary and secondaryendpoints of this study were clinical pregnancy and delivery of a liveborn infant, respectively.

-   Statistical Analysis

A statistical analysis was performed using StatView, version 5 (SASInstitute Inc., Cary, N.C.). Continuous variables were analyzed byWilcoxon signed rank test. Categorical variables were analyzed by χ²analysis or Fisher's exact test as indicated. A probability of <0.05 wasconsidered to be statistically different.

Results

-   Study Population

Age, obstetrical and infertility histories of the study and controlgroups are not different between the treatment group and controls (Table1). The average age of the treatment group was 36.2±2.5 (mean±SD) years,which was comparable to that of control (36.1±4.1). Indications forassisted reproductive technology (ART) treatment of both groups werecomparable. About one-fourth of the patients in the treatment group hada history of a positive pregnancy test, but none delivered a live borninfant. The same trend was seen in the control group. The mean numbersand standard deviation (SD) of previous ET attempts, transferred embryosand transferred MGEs in the treatment group were 5.8±2.9, 8.6±5.8, and5.0±2.1, respectively, and these were not significantly different fromthose in the control group (5.9±2.7, 7.4±3.4 and 4.9±2.0, respectively).

-   TH1/Th2 Cell Ratios

The results of the analyses of the Th1 and Th2 cells are also shown intable 1. The percentages of IFN-γ producing CD4+T lymphocytes (Thl) inthe treatment and control groups were 27.7±9.4 and 26.7±7.3 (mean ±S.D.)respectively, which revealed no significant differences. The percentagesof IL-4 producing CD4⁺ T lymphocytes (Th2) in the treatment and controlgroups were 1.8±0.6 and 1.7±0.6 respectively, which was notsignificantly different. The Th1/Th2 cell ratio in the treatment groupwas 16.1±7.0, which was similar to that in the control group (16.7±5.2).

-   Treatment Outcome

The ART outcomes in both groups are summarized in table 2. The numbersof the transferred embryos per cycle in the treatment and control groupswere 1.4±0.5 and 1.4±0.5, respectively (P=NS). The percentage of MGEswas 68.9% in the treatment group, which was comparable to that in thecontrol group (70.8%). No one achieved a pregnancy in the control groupand 16 patients out of 25 (64%) in the treatment group had a positivepregnancy test and progressed to clinical pregnancies with tacrolimustreatment. There was no biochemical loss and the implantation rate was45.7% (16/35). The number of patients treated with 1 mg, 2 mg, or 3 mgof tracrolimus were 12, 8, and 5, respectively (table 3). The clinicalpregnancy and live birth rates in the patients treated with 1 mg oftacrolimus were 83.3 and 83.3%, respectively, and these rates in thepatients with 2mg and 3 mg of tacrolimus were 50.0 and 37.5%, and 40.0and 40.0%, respectively (Table 3).

Among the pregnant patients in the treatment group, only one experienceda miscarriage (6.3%) at 10 weeks gestation. Total fifteen healthy babieswere born with tacrolimus treatment and the live birth rate in thetreatment group was 60.0%. Obstetrical outcome of the newborns were asfollows; birth weight (mean±SD) was 2,995±400 g, mean gestational daysto delivery was 279.5±10.6, APGAR scores of 1 and 5 minutes after birthwere 8.3±0.5 and 9.2±0.5, respectively, and 6 babies were delivered byCaesarian section and 9 were born by vaginal delivery (Table 4).

With tacrolimus treatment, twelve of 16 women (75%) who receivedfrozen—thawed ET and 4 of 9 (44.4%) women with fresh ET become pregnant.Frozen-thawed ET cycle had a higher success rate than fresh IVF-ET cyclehowever, it did not reach statistical significance. Twelve of 16 women(75%) with FET and 3 of 9 (33.3%) women with fresh ET have delivered alive born infant. There is a trend of increased success rate in FETcycle as compared with fresh ET cycle in RIF patients with tacrolimustreatment (P<0.1).

Discussion

Repeated implantation failures (RIF) is often determined when embryos ofgood quality fail to implant following at least 3 consecutive IVFattempts, in which 1-2 embryos of high grade quality are transferred ineach cycle.¹⁵ However there is no general consensus for a definitionyet. Implantation involves maternal immune system and many mediatorssuch as cytokines, chemokines and various growth factors from an embryoand endometrium. Hence, stratifying patients only based on theirinfertility history, such as RIF may not be adequate for furtherevaluation and treatment. A recent review of endometrial receptivityreport that there is no single biomarker which is specific forendometrial receptivity.¹⁶ Although we measured endometrial thickness inthis study as a standard infertility work up, objective diagnosis ofendometrial receptivity is seemingly remote. The same may hold true forimmunological factors, since only Th1/Th2 cell ratios are investigatedin this study.

The treatment modalities for RIF patients with Th1/Th2 elevation havebeen previously reported. High-dose intravenous gammaglobulin G (IVIG)has been reported to be effective in women with repeated IVF andimplantation failures.¹⁷⁻¹⁹ IVIg reduces Th1/Th2 lymphocyte ratio inperipheral blood, which confirms a significance of Th1/Th2 immuneregulation in implantation and maintenance of pregnancy.¹⁷ Etanercept(Enbrel®), recombinant human TNF receptor [p75]:Fc fusion protein, hasbeen reported to be effective in RIF patients who had an elevatedperipheral blood Th1/Th2 ratio.²⁰ A recent publication detailing the useof IVIg and adalimumab (Humira™), which is a recombinant human IgG1monoclonal antibody specific for TNF, showed improved pregnancy rates inRIF patients accompanied by Th1/Th2 elevation.²¹ However, the safety ofanti-TNF drugs, such as etanercept or adalimumab has not beenestablished yet for either infertile or pregnant women.²² Sincetacrolimus has accumulated pregnancy safety data from transplantpopulations, in this study, we investigated if tacrolimus is suitable toregulate immunological responses and improve reproductive outcomes inwomen who had an extreme number of RIF (5 or more) after IVF cycles.

According to a previous report, a significant proportion of RIF patientshave a dysregulated cellular immune effector mechanism^(4,23) and anincreased Thl cytokine response was reported in women with RIF duringtheir failed ART cycle.²⁴ In this study, 51.9% of women with RIF (≥5)had increased Th1/Th2 cell ratios particularly, when IL-4 and IFN-γproducing T cells were investigated. This was consistent with previousstudies. ^(4,23)

Presence of Th1 immunity to semi-allogeneic embryo mirrors a graftrejection process during organ transplantation. Immunosuppressive drugs,such as tacrolimus are often prescribed for the recipients of anallogeneic organ transplant in order to reduce the risk of organrejection.

Tacrolimus inhibits both T-lymphocyte signal transduction and IL-2transcription.²⁰ In this study, Th1 shift was detected by measuringCD4+/IFN-γ and CD4+/IL-4 positive cell ratios. It has been reported thatsupra-physiological dose of IFN-γ induces peri-implantation failures inanimal model by decreasing Treg and Th17 cells locally at implantationsite.²⁵ Additionally, insulin-like growth factor binding protein 7(IGFBP7) can lead to IFN-γ up-regulation with concurrent down regulationof IL-4 and IL-10, which lead to implantation failures in mice model.²⁶Contrarily, IFN-γ induces appropriate arterial modifications at theimplantation site.²⁷ Therefore, adequate expression and regulation ofIFN-γ plays a key role in successful implantation and pregnancy.Considering dose dependent contradictory biological effects of IFN-γ,tracrolimus dose was determined based on Th1/Th2 cell ratio of womenwith RIF in this study. In addition, tacrolimus dose for studypopulation was ranged 20-60% of prophylactic renal transplant schedule(0.1 mg/kg/day). All patients tolerated tacrolimus treatment well and noone reported side effect while on tacrolimus treatment. However, thisobservation should be carefully interpreted since the study size issmall.

Tacrolimus has been utilized throughout pregnancy for women who havereceived an allogeneic organ transplant, and many female recipients havegiven birth while taking tacrolium.²⁸ Tacrolimus reduces peptidyl-prolylisomerase activity by binding to the immunophilin FKBP12 (FK506 bindingprotein) creating a new complex. This FKBP12-FK506 complex interactswith and inhibits calcineurin thus inhibiting both T-lymphocyte signaltransduction and IL-2 transcription.²⁹ Although this activity is similarto cyclosporin, studies have shown that the incidence of acute rejectionis reduced by tacrolimus use over cyclosporin.³⁰ The effects onshort-term immunosuppression and graft survival in patients are found tobe similar between the two drugs, however, tacrolimus results in a morefavorable lipid profile which may have important long-term implicationsgiven the prognostic influence of rejection on graft survival.³¹

Tacrolimus is classified as class C drug by the FDA pregnancy category.When considering RIF is not a life-threatening disease, class C drugapplication for women undergoing IVF cycle should be determined aftercareful benefit risk assessment. The safety of tacrolimus for bothmother and fetus/baby during pregnancy has been well established in manyreports of female transplant recipients who achieved a post-transplantpregnancy.^(8,32) In 236 babies born from the kidney transplantrecipients with tacrolimus exposure during pregnancy, the still bornrate was 2% and the neonatal death rate was 2%.³³ Other reportedobstetrical complications were preeclampsia 32%, diabetes duringpregnancy 8%, hypertension 56%, infection 22%, and spontaneous abortion26%.³³ In the present study, 60% (n=15) of the RIF patients with aTh1/Th2 elevation could achieve pregnancy and delivered a live borninfant. No one had obstetrical complication and only one baby showedtransient tachypnea of newborn. Differences in obstetrical outcomesbetween our study population and the renal transplant population couldbe related to the differences in general health status of the studypopulation, dosage of tacrolimus and a duration of drug exposure. Thesepatients could not achieve pregnancy prior even after ET of 5 MGEs.

Tacrolimus treatment significantly increased implantation rate, clinicalpregnancy rate and live birth rate in women with RIF and elevatedTh1/Th2 cell ratios. According to our data, the benefit of tacrolimustreatment during implantation period overweighs the risk to thedeveloping fetus. Evaluation of Th1/Th2 cell ratio can be utilized as abiomarker for a selection of women with RIF, who will respond immunesuppression treatment by tacrolimus and have successful reproductiveoutcome.

In this study, patients who had FET had higher success rate (75%) thanfresh ET (33.3%) while on tacrolimus treatment. Recently, freeze-allstrategies have been rapidly adopted by IVF centers since more receptiveendometrium is expected in FET cycle, increased health of children bornfrom the frozen-thawed embryos compared with those from fresh ET cycle,and increased success rates of FET that now almost equal the successrates of fresh ET.²⁷ Per 2011 CDC report, live birth rate of fresh ETcycle was 40% and FET was 39% for women under the age of 35 years.³³ Inthis study, despite of higher age of study population (mean age 36), FETsuccess rate was 75% which was 1.9 times higher than reported successrate (39%) of women under 35.²⁷ Therefor, FET cycle with tacrolimustreatment can be applicable in RIF patients with advanced age group(>35). High clinical pregnancy rate of FET cycle may be resulted from acombined effect of Th1 immune regulation by Tacrolimus and morereceptive endometrium by FET cycle. This study, however, has alimitation, since this is not a randomized controlled trial and a samplesize is small. Moreover, endometrial changes or peripheral immuneresponses after tacrolimus treatment were not evaluated thoroughly.Further study is needed for tacrolimus effect on systematic immuneresponses and endometrial receptivity. In conclusion, animmunosuppressive treatment using tacrolimus improved pregnancy outcomesin RIF patients with elevated Th1/Th2 ratios. Further investigation,taking into account of Th17 and T regulatory cells, and endometrialreceptivity is needed in considerable detail. Conclusively, this studyindicates tacrolimus treatment significantly increases clinicalpregnancy rate and live birth rate in RIF patients with shifted Th1immune responses without increased maternal severe complications.

EXAMPLES

TABLE 1 Age, obstetrical and infertility histories and clinicalcharacteristics of women with repeated implantation failures (5 or more)and elevated Th1/Th2 ratios who were treated with tacrolimus (treatmentgroup) and without tacrolimus (control group). Treatment Control groupgroup P Characteristics (n = 25) (n = 17) value Age (years) 36.2 ± 2.5 36.1 ± 4.1  ns Body mass index (Kg/m²)^(a) 20.2 ± 1.4  20.4 ± 1.3  nsIndications for ART^(a), % (n) ns Unexplained infertility 80.0 (20)  100 (17) Male infertility 16.0 (4)  0 Tubal infertility 4.0 (1) 0Previous pregnancy history, % (n) ns No 72.0 (18)  64.7 (11) Yes 28.0(7)  35.3 (6) Obstetrical history, % (n) ns Gravidity ≥ 1 28.0 (7)  35.3(6) Parity ≥ 1   0 (0)   0 (0) SAB^(b) ≥ 1 28.0 (7)  35.3 (6) Previousembryo transfer history Number of failed embryo transfer 5.8 ± 2.9 5.9 ±2.7 ns cycle (n) Total number of transferred embryos 8.6 ± 5.8 7.4 ± 3.4ns (n) Total number of transferred MGE^(c) 5.0 ± 2.1 4.9 ± 2.0 ns (n)sAnalyses of the Th1 and Th2 cells Th1 cells^(d) (%) 27.7 ± 9.4  26.7 ±7.3  ns Th2 cells^(e) (%) 1.8 ± 0.6 1.7 ± 0.6 ns Th1/Th2 ratio 16.1 ±7.0  16.7 ± 5.2  ns Women who miscarried in a former IVF cycle or hadIVF cycles, or received vaccination in 3 months were excluded in thestudy. Values are mean ± standard deviation unless otherwise specified.^(a)ART; assisted reproductive technology ^(b)SAB; spontaneous abortion.Women who miscarried within 3 months from prior IVF cycle were excludedfrom the study. ^(c)MGEs; morphologically good-quality embryos ^(d)Th1cell; Interferon gamma producing T helper cell (CD3⁺/4⁺/IFN-γ⁺) ^(e)Th2cell; IL-4 producing T helper cell (CD3⁺/4⁺/IL-4⁺)

Table 1 shows the age, obstetrical history, infertility history, andclinical characteristics of women with repeated implantation failures (5or more times) and an elevated Th1/Th2 cell ratios, who were treatedwith tacrolimus (treatment group), and who were not treated (controlgroup). In the table, “Body mass index” means BMI, “Unexplainedinfertility” means sterility of unknown causes, “Male infertility” meansmale sterility, and “Tubal infertility” means tubal sterility. “Previouspregnancy history” means the presence (Yes) or absence (No) of apregnancy history in the past; “Obstetrical history” means a history ofchildbirth (Gravidity=reproductive history, Parity=birth history,SAB=spontaneous miscarriage); “Previous embryo transfer history” means ahistory of embryo transplantation in the past; “Number of failed embryotransfer cycle (n)” means the number of failed embryo transplantationcycles; “Total number of transferred embryos (n)” means the total numberof transplanted embryos; and “Total number of transferred MGEs (n)”means the total number of transplanted MGEs. Th1/Th2 ratio refers to theTh1/Th2 cell ratio.

TABLE 2 Reproductive outcomes of women with repeated implantationfailures who were treated with tacrolimus (treatment group) and withouttreatment (control group). Treatment Control group group P (n = 25) (n =17) value Number of transferred cycles, n 25 17 — Number of transferredembryos  1.4 ± 0.5  1.4 ± 0.5 ns per cycle Frozen-thawed embryotransfer, n 16 11 — Fresh embryo transfer, n 9 6 — Percentage ofMGEs^(a) (%) 68.9 70.8 ns Endometrial lining (mm) 10.8 ± 1.4 11.0 ± 1.9ns Positive hCG (%) 64.0 0 P < 0.0001 Implantation rate (%) 45.7 0 P <0.0001 Biochemical pregnancy rate (%) 0 0 — Clinical pregnancy (n) 16 0— Clinical pregnancy of fresh ET (%) 44.4 0 ns Clinical pregnancy ofcryo-thaw 75.0 0 P < 0.0001 ET (%) Clinical pregnancy rate per ET (%)64.0 0 P < 0.0001 Spontaneous abortion rate (%) 6.3 ns Live birth (n) 150 — Live birth rate of fresh ET (%) 33.3 0 ns Live birth rate of thawedET (%) 75.0 0 P < 0.0001 Live birth rate (%) 60.0 0 P < 0.0001 Livebirth (n) 15 0 — RIF; repeated implantation failure, ^(a)MGEs:morphologically good embryo Values are mean ± standard deviation unlessotherwise specified.

Table 2 shows the birth outcome of women with repeated implantationfailures (RIF), who were treated with tacrolimus (treatment group) andwho were not treated (control group). In the table, “Number oftransferred cycles” means the number of transplantation cycles; “Numberof transferred embryos per cycle” means the number of transplantedembryos per cycle; “Frozen-thawed embryo transfer, n” means the numberof transplantations of frozen and thawed embryos; “Fresh embryotransfer, n” means the number of transplantations of fresh embryos;“Percentage of MGEs” means the proportion (%) of MGEs; “Endometriallining” means the thickness of the endometrium; “Positive hCG (%)” meansthe proportion (%) of positive hCG; “Implantation rate (%)” means theratio of implantation (%); “Biochemical pregnancy rate (%)” means thebiochemical pregnancy ratio (%); “Clinical pregnancy (n)” means thenumber of individuals who have achieved clinical pregnancy; “Clinicalpregnancy of fresh ET (%)” means the clinical pregnancy ratio (%) withfresh ET; “Clinical pregnancy rate per ET (%)” means the clinicalpregnancy ratio (%) per round of ET; “Spontaneous abortion rate” meansthe ratio of spontaneous miscarriage (%); “Live birth (n)” means thenumber of live births; “Live birth rate of fresh ET (%)” means the livebirth ratio (%) with fresh ET; “Live birth rate of thawed ET (%)” meansthe live birth ratio (%) with frozen-thawed ET; and “Live birth rate(%)” means the live birth ratio (%).

TABLE 3 Reproductive outcomes of women with repeated implantationfailures who were treated with various dosage of tacrolimus (treatmentgroup). Treatment group (n = 25) Treatment dosage Number of patientstreated with 1 mg of tacrolimus, n 12 518 2 mg of tacrolimus, n 8 519 3mg of tacrolimus, n 5 520 Clinical pregnancy (n) 16 with 1 mg oftacrolimus (%) 83.3 With 2 mg of tacrolimus 50.0 with 3 mg of tacrolimus40.0 Live birth (n) 15 with 1 mg of tacrolimus (%) 83.3 With 2 mg oftacrolimus 37.5 with 3 mg of tacrolimus 40.0

Table 3 shows the birth outcome of women with repeated implantationfailure (RIF) who were treated with various treatment dosages oftacrolimus (treatment group). In the table, “Number of patients treatedwith” means the number of patients who were treated with a predetermineddose; “Clinical pregnancy (n)” means the number of individuals who haveachieved clinical pregnancy; and “Live birth (n)” means the number oflive births. The dosage of tacrolimus is 1 mg, 2 mg, or 3 mg.

TABLE 4 Obstetrical and neonatal outcome of women with multipleimplantation failures (5 or more) who were treated with tacrorimus.Number of newborns (n) 15 Mode of delivery vaginal delivery (n) 9Cesarean section (n) 6 Gestational days to delivery (days) 279.5 ± 10.6 Birth weight (gm)^(a) 3,021 ± 371   Height (cm)^(a) 50.9 ± 0.9  Sex ofnewborn, boy/girl (n/n) 7/8 APGAR score at 1 minutes after birth 8.3 ±0.5 APGAR score at 5 minutes after birth 9.2 ± 0.6 Umbilical cord gasanalysis (pH)  7.2 ± 0.05 Umbilical cord length (cm) 56.2 ± 12.2Obstetrical complications (n) 0 Neonatal complications (n) One case ofTTN 1 case Values are mean ± standard deviation unless otherwisespecified. ^(a)TTN; transient tachypnea of newborn.

Table 4 shows the outcome of births and newborn baby births of womenwith multiple implantation failures (5 or more times), who were treatedwith tacrolimus. In the table, “Number of newborns (n)” means the numberof newborn babies; “Mode of delivery” means whether the mode of deliveryis vaginal delivery or Cesarean section; “Gestational days to delivery(days)” means the number of gestational days; “Birth weight” means thebody weight at the time of birth; “Height” means the height at the timeof birth; “Sex of newborn” means whether the gender of the newborn babyis male (boy) or female (girl); “APGAR score at 1 (or 5) minutes afterbirth” means the APGAR score obtained one minute (5 minutes) afterbirth; “Umbilical cord gas analysis (pH)” means the result of a gasanalysis (pH) of the umbilical cord; “Umbilical cord length” means thelength of the umbilical cord; “Obstetrical complications (n)” means thenumber of individuals with onset of pregnancy complications; and“Neonatal complications (n)” means that there were instances of onset oftransient tachypnea of the newborn (TNN).

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Example 2 Materials and Methods Subject Group for Research

Among women who were capable of IVF/ET and had a history of three ormore consecutive miscarriages, stillbirths, or intrauterine fetal growthretardations, eleven infertilitas patients who could not bear child eventhough they received one or more times of existing treatment, wereselected as subjects. These patients were a group of patients for whomclear findings of abnormality related to infertility were not recognizedwith a serological examination that had been performed in advance.

The patients were evaluated through a transvaginal ultrasoundexamination, a Fallopian tube patency test, and hysteroscopy before anindex ART cycle. Among the participants, no one had submucosal myoma,endometrial polyps, intrauterine adhesion, congenital abnormalities inthe uterus, or hydrosalpinx. Also, any pregnant women, or any womenexhibiting chronic medical symptoms or inflammatory symptoms wereexcluded from the subjects. Any women who had experienced miscarriage inthe previous IVF cycle, who had received an IVF cycle, or who hadreceived vaccination, all within three months, were excluded from thesubjects. Any women with acquired or congenital thrombophilia wereexcluded from the subjects.

Analysis of Th1 Cells and Th2 Cells

The analysis was carried out according to the method described inExample 1. However, the determination of the Th1/Th2 cell ratio in apatient was carried out immediately after establishment of pregnancy(the day of confirming establishment of pregnancy). The level of theTh1/Th2 cell ratio was determined according to the average value +1standard deviation method in the same manner as in Example 1, and aTh1/Th2 cell ratio that was equal to 10.3 or higher than that wasclassified as an increased Th1/Th2 cell ratio.

Treatment with Tacrolimus

All of the eleven patients were subjected to treatment with tacrolimusin the same manner as in Example 1, except for the duration ofadministration. The dose of tacrolimus was 1 mg per day for patients whohad a Th1/Th2 cell ratio of 10.3 or higher and lower than 13.0 (fourpatients); the dose was 2 mg per day for patients who had a Th1/Th2 cellratio of 13.0 or higher and lower than 15.8 (three patients); and thedose was 3 mg per day for patients who had a Th1/Th2 cell ratio of 15.8or higher (four patients). The duration of administration of tacrolimuswas from the time point immediately after establishment of pregnancy wasconfirmed by a pregnancy test and the Thl/Th2 cell ratio was checked,until the day of delivery, in all cases.

IVF-ET Treatment

The treatment was carried out in the same manner as in Example 1.

Statistical Analysis

The analysis was carried out in the same manner as in Example 1.

Results

The features of the patients who received the treatment are summarizedin Table 5.

The results of the treatment are summarized in Table 6. Eight out of theeleven patients who received the treatment obtained healthy babies byfull term birth. Premature birth case 1 disclosed herein is a case witha history of 10 or more times of miscarriage due to resistance to allthe therapeutic methods that are currently implemented, as described inthe Background Art. Other therapeutic methods were not used incombination, and childbearing was achieved by the treatment withtacrolimus alone.

TABLE 5 Treatment group (n = 11) Age (years) 37.3 ± 3.6  Medical historyof pregnancy % (n) No 0 (0) Yes 100 (11)  History of pregnancy Number oftimes of pregnancy 4.8 ± 2.5 Number of times of childbearing (live bornbaby) 0.2 ± 0.4

TABLE 6 Mis- Full term carriage Premature birth birth Number of newbornbabies 2 1 8 (n) Mode of delivery Vaginal delivery (n) — 0 8 Cesareansection (n) — 1 0 Number of gestational days — 208 279.3 ± 6.0  (days)Birth weight (gm) — 748 3149 ± 234  Gender of baby male/female — 0/1 4/4(n/n) APGAR Score 1 minute — 6 8.0 ± 1.0 APGAR Score 5 minute — 8 9.0 ±0.5 Umbilical cord blood pH — 7.304  7.3 ± 0.12 Length of umbilical cord(cm) — 27.5 62.4 ± 12.9 Complications of mother — 1 (Pregnancy- 0induced hypertension) Complications of newborn — 1 (Low birth 0 babyweight baby)

Example 3 Materials and Methods Subject Group for Research

Women who were capable of IVF/ET, and had a history of havingpregnancy-induced hypertension syndrome during the previous gestation,or showed a clinical finding of being susceptible to the development ofpregnancy-induced hypertension syndrome, were selected as subjects. Oncepregnancy-induced hypertension syndrome develops, the possibility ofexperiencing repeated onset in the subsequent gestations is very high.

The patients were evaluated through a transvaginal ultrasoundexamination, a Fallopian tube patency test, and hysteroscopy before anindex ART cycle. Any pregnant women, or any women exhibiting chronicmedical symptoms or inflammatory symptoms were excluded from thesubjects. Any women who had experienced miscarriage in the previous IVFcycle, who had received an IVF cycle, or who had received vaccination,all within three months, were excluded from the subjects. Any women withacquired or congenital thrombophilia were excluded from the subjects.

Treatment With Tacrolimus

All the patients were subjected to treatment with tacrolimus in the samemanner as in Example 2. The dose of tacrolimus was 1 mg to 3 mg per day.The duration of administration of tacrolimus was from the time pointimmediately after establishment of pregnancy was confirmed by apregnancy test, until the day of delivery, in all cases. In Case 1 andCase 3 described below, 3 mg of tacrolimus per day was administeredeveryday from the time point immediately after establishment ofpregnancy was confirmed and until the second trimester of pregnancy, and2 mg of tacrolimus per day was administered everyday from the thirdtrimester of pregnancy until the delivery. In Case 2 described below, 2mg of tacrolimus per day was administered everyday from the time pointimmediately after establishment of pregnancy was confirmed and until thedelivery.

IVF-ET treatment

The treatment was carried out in the same manner as in Example 2.

Statistical Analysis

The analysis was carried out in the same manner as in Example 2.

Confirmation of Results

The effects of administering tacrolimus were evaluated on the basis ofwhether the patients satisfactorily maintained the state of pregnancywithout developing pregnancy-induced hypertension syndrome atpredetermined time points between the second trimester and the thirdtrimester of pregnancy. Cases in which the evaluation has been roughlycompleted are as follows.

In a case in which intrauterine fetal death occurred in week 38 ofpregnancy due to pregnancy-induced hypertension syndrome in the previousgestation (Case 1), the patient did not experience an increase in bloodpressure as a result of administration of tacrolimus and gave birth to ahealthy baby in week 39. In a case of twin pregnancy with infertilitasand positive anti-phosphorus antibody, with a high potential ofdeveloping pregnancy-induced hypertension syndrome (Case 2), the patientdid not experience an increase in blood pressure as a result ofadministration of tacrolimus and gave birth to a healthy baby in week 35(premature rupture of the membrane caused by cough, premature birth oftwin). Furthermore, in a case in which the patient had edema from week25, experienced an increase in blood pressure from week 28, wasdiagnosed as pregnancy-induced hypertension syndrome, and sufferedintrauterine fetal death in week 32 in the previous gestation (Case 3),the patient is currently passing week 34 of pregnancy auspiciouslywithout edema and without a tendency of blood pressure increase, as aresult of administration of tacrolimus.

During the progress of pregnancy, babies grew satisfactorily in allcases, and abnormalities were not observed in both the mother and thechild.

The present invention is not intended to be limited to the variousembodiments and the various Examples described above, variousmodifications can be made to the extent that is disclosed in the claims,and embodiments obtainable by appropriately combining the technicalmeans respectively disclosed in different embodiments and differentExamples are also included in the technical scope of the presentinvention. Furthermore, new technical features can be formed bycombining the technical means respectively disclosed in the variousembodiments and the various Examples.

1. A medicine for ameliorating pregnancy conditions, the medicinecomprising a compound represented by the following General Formula (I)or a pharmaceutically acceptable salt thereof as an active ingredient:

wherein each of the adjacent pairs of R¹ with R², R³ with R⁴, and R⁵with R⁶ independently (a) represents two adjacent hydrogen atoms, or R²may be an alkyl group, or (b) may form another bond between the carbonatoms to which the pair members are respectively bonded; R⁷ represents ahydrogen atom, a hydroxyl group, or a protected hydroxyl group, or maybe bonded to an alkyl and together represent an oxo group; R⁸ and R⁹each independently represent a hydrogen atom or a hydroxyl group; R¹⁰represents a hydrogen atom, an alkyl group, an alkyl group substitutedwith one or more hydroxyl groups, an alkenyl group, an alkenyl groupsubstituted with one or more hydroxyl groups, or an alkyl groupsubstituted with an oxo group; X represents an oxo group, (a hydrogenatom, a hydroxyl group), (a hydrogen atom, a hydrogen atom), or a grouprepresented by formula: —CH₂O—; Y represents an oxo group, (a hydrogenatom, a hydroxyl group), (a hydrogen atom, a hydrogen atom), or a grouprepresented by formula: N—NR¹¹R¹² or formula: N—OR¹³; R¹¹ and R¹² eachindependently represent a hydrogen atom, an alkyl group, an aryl group,or a tosyl group; R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²², and R²³ eachindependently represent a hydrogen atom or an alkyl group; R²⁴represents a ring which can include one or more heteroatoms and may besubstituted as desired; and n represents 1 or 2, and in addition to themeanings described above, Y, R¹⁰ and R²³ may also be bonded togetherwith the carbon atoms to which Y, R¹⁰ and R²³ are bonded, and representa heterocyclic group formed from a saturated or unsaturated 5-memberedor 6-membered ring and containing a nitrogen atom, a sulfur atom, and/oran oxygen atom, and the heterocyclic group may be substituted with oneor more groups selected from an alkyl group, a hydroxyl group, analkyloxy group, a benzyl group, a group represented by formula:—CH₂Se(C₆H₅), and an alkyl group substituted with one or more hydroxylgroups.
 2. The medicine according to claim 1, wherein the compound istacrolimus or a pharmaceutic ally acceptable salt thereof.
 3. Themedicine according to claim 1, wherein the medicine amelioratesdefective pregnancy conditions attributed to immunologicalabnormalities.
 4. The medicine according to claim 1, wherein themedicine ameliorates at least one defective pregnancy condition selectedfrom the group consisting of sterility, infertility, andpregnancy-induced hypertension syndrome.
 5. The medicine according toclaim 1, wherein the medicine is administered to a subject of treatmentat a dose of 5 mg or less per day as the amount of the activeingredient.
 6. The medicine according to claim 5, wherein the amount ofthe active ingredient is in the range of from 1 mg to 3 mg per day. 7.The medicine according to claim 1, wherein the medicine is administeredto a subject having an increased Th1/Th2 cell ratio compared to a normalperson.
 8. The medicine according to claim 1, wherein the medicine isadministered to a subject who is subjected to transplantation of anembryo obtained by in vitro fertilization.
 9. The medicine according toclaim 1, wherein in a case in which the medicine is intended forsterility as a defective pregnancy condition, the medicine isadministered at least during a period from 1 day before or 2 days beforethe implantation of a fertilized ovum or a transplanted embryo into theendometrium, to 0 days after the implantation; and in a case in whichthe medicine is intended for a defective pregnancy condition other thansterility, the medicine is administered at least for a period from theestablishment to the 200th day of pregnancy.